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hydroxyapatite hydrogels enhance angiogenesis in in-ovo experiments
Journal Pre-proofs Thyroxine-loaded chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels enhance angiogenesis in ex-ovo experiments Muhammad Hamza Malik, Lubna Shahzadi, Razia Batool, Sher Zaman Safi, Abdul Samad Khan, Ather Farooq Khan, Aqif Anwar Chaudhry, Ihtesham Ur Rehman, Muhammad Yar PII: DOI: Reference:
S0141-8130(19)34614-8 https://doi.org/10.1016/j.ijbiomac.2019.10.043 BIOMAC 13492
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
19 June 2019 25 September 2019 3 October 2019
Please cite this article as: M. Hamza Malik, L. Shahzadi, R. Batool, S. Zaman Safi, A. Samad Khan, A. Farooq Khan, A. Anwar Chaudhry, I. Ur Rehman, M. Yar, Thyroxine-loaded chitosan/carboxymethyl cellulose/ hydroxyapatite hydrogels enhance angiogenesis in ex-ovo experiments, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.043
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Thyroxine-loaded chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels enhance angiogenesis in ex-ovo experiments Muhammad Hamza Malika, Lubna Shahzadia, Razia Batoola, Sher Zaman Safia, Abdul Samad Khana,c, Ather Farooq Khana, Aqif Anwar Chaudhrya, Ihtesham Ur Rehmana,b, Muhammad Yara,* a
Interdisciplinary Research Center in Biomedical Materials, COMSATS University Islamabad, Lahore Campus,54000, Pakistan b Engineering Department, Lancaster University, Lancaster, UK c Department of Restorative Dental Sciences, College of Dentistry, University of Dammam,31441, Saudi Arabia
*Correspondence Address: a
Interdisciplinary Research Center in Biomedical Materials, COMSATS University Islamabad, Lahore Campus,54000, Pakistan; Tel: 0092 42 001007 ext 829; Fax: 0092 42 35321090; E-mail: [email protected] (M. Yar)
Abstract Angiogenesis is one of the most important processes in repair and regeneration of many tissues and organs. Blood vessel formation also play a major role in repair of dental tissue(s) after ailments like
periodontitis.
Here
we
report
the
preparation
of
chitosan/carboxymethyl
cellulose/hydroxyapatite based hydrogels, loaded with variable concentrations of thyroxin i.e., 0.1 μg/ml, 0.5 μg/ml and 1 μg/ml. Scanning electron microcopy images (SEM) showed all hydrogels
were found to be porous and solution absorption study exhibited high swelling potential in aqueous media. FTIR spectra confirmed that the used materials did not change their chemical identity in synthesized hydrogels. The synthesized hydrogels demonstrated good bending, folding, rolling and stretching abilities. The hydrogels were tested in chick chorioallantoic membrane (CAM) assay to investigate their angiogenic potential. Hydrogel containing 0.1 μg/ml of thyroxine showed maximum neovascularization. For cytotoxicity analyses, preosteoblast cells (MC3T3-E1) were seeded on these hydrogels and materials were found to be non-toxic. These hydrogels with proangiogenic activity possess great potential to be used for periodontal regeneration. Key words: Tissue engineering, periodontal regeneration, thyroxine, angiogenesis, chitosan, carboxymethyl cellulose, hydroxyapatite, injectable gels Introduction 1
Tissue engineering has gained popularity over years as a potential solution in the field of regenerative medicine; whereby natural, semisynthetic and synthetic materials having medical response are implanted to mimic the natural response of the failed tissue or organ[1, 2]. Angiogenesis has been one of the determining factors for the regeneration of different tissues and organs in human body. It is a vital process in wound healing, as newly formed blood vessels helps to form the granulation tissue and provide oxygen and nutrition to the injured area [3]. It is also one of the essential requirements for hard tissue regeneration. A number of research reports have expressed the importance of angiogenesis in healing and repair of fractured bone [4], e.g. Schmidt et. al., found that blood vessel formation occurs before new bone formation in rabbits. [5]. Destruction of periodontal tissue is termed as periodontal disease and characterized by the necrosis of gingiva, alveolar bone, periodontal ligament and cementum [6]. According to Center for Disease Control and Prevention, 47.8% of the adults aged 30 or above were affected by some form of periodontal disease in USA, 8.7% having mild, 30.0% having moderate, and 8.5% with severe form of periodontitis [7]. The prevalence of periodontitis has been found to be 64% in older adults, aged 65 years and older in US [7]. Generally, scaling and curettage, medications including mouthwashes, antibiotics and surgical interventions have been employed to treat periodontal diseases [8], however little has been achieved to completely reverse the disease progression and regenerate the damaged periodontal tissue. Periodontal disease has been related to other systemic diseases and disorders as well, like adverse events in pregnancy [9], oral cancer [10] and diabetes [11], which shows the need to form new interventions to permanently treat the disease. Periodontium is richly supplied with blood vessels and induction of angiogenesis is necessary in order to supply nutrients and oxygen to the grafted tissue engineered construct for better repair [12]. Another study demonstrated that enamel matrix derivative induces angiogenesis during periodontal wound healing [13]. In addition to periodontium, angiogenesis has been found to be essential in the regeneration of pulp [14, 15], which results in the production of dentin-like tissue along the dentinal wall by continuous layer of odontoblast –like cells [16]. Growth factors that are generally involved in angiogenesis are found to be up-regulated in periodontal repair process. The fibroblast growth factor (bFGF) promotes the cell growth of periodontal ligament and up-regulates the laminin mRNA level, which further promotes the process of angiogenesis [15, 17].
2
Thyroxine is one of the vital hormones with various physiological functions within human body. One of the functions, associated with thyroxine is its ability to induce angiogenesis by several mechanisms [18]. Mainly, thyroxine causes the transcription of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), the two main growth factors responsible for blood vessel growth by activating integrin αvβ3 [19]. A number of polymeric biomaterials such as chitosan, polycaprolactone and hydroxyethyl cellulose have been shown to possess potential for periodontal treatment by helping to regenerate the tissue and/or delivering drugs [20]. Chitosan is a natural copolymer comprising of glucosamine and N-acetylglucosamine, generally derived from crustacean shells by partial deacetylation of chitin [6] and it has been used alone or with other polymers [21-23] as a biomaterial for its attributes of being biocompatible, biodegradable and bacteriostatic [18, 24, 25]. Many research groups have shown potential of chitosan for periodontal treatment and regeneration [26]. Hydroxyapatite (HA), a naturally occurring bioactive material, has been incorporated in chitosan by wet chemical method [27, 28] or lyophilization [29]. Carboxymethyl cellulose is another polymer with well-known use in food and pharmaceutical industry. Due to its slow degradability and biocompatibility, it has been used for hard tissue regeneration along with chitosan and HAp [30, 31]. Hydroxyapatite is a naturally occurring material, responsible for the hardness of bone, enamel and dentine [31, 32]. Since there is absolute need for biocompatible membranes which can support angiogenesis and at the same time help tissue regeneration. In current research, thyroxine loaded biocompatible proangiogenic hydrogels have been developed from chitosan, carboxymethyl cellulose and hydroxyapatite. The synthesized membranes showed good foldability and stretching properties and upon supplying slight liquid these membranes can be converted into injectable hydrogels. CAM assay showed the angiogenic activity of the synthesized materials and variable amounts of thyroxin were tested to find the suitable concentration required for efficient angiogenesis. 2- Materials and Methods 2.1- Materials All chemicals used in this study were analytical grade. Chitosan (CS) was purchased from Mian Scientific Company (Lahore, Pakistan) and further purified as we described earlier [32] (Mw: 3
144,105.g/mol., degree of deacetylation (DD): 83%, intrinsic viscosity: 30.85 ml/g). HAp was also synthesized in our laboratory [33]. Glacial acetic acid (BDH Laboratory Supplies UK) was of analytical grade and was used without further purification. Thyroxine was provided by GlaxoSmithKline Pakistan. Mouse pre-osteoblast cell line MC3T3-E1 sub-clone 14 was purchased from ATCC cell bank (USA). 2.2- Preparation of freeze-dried hydrogels Chitosan (2% w/v) was dissolved in 1% acetic acid solution and the thyroxine solution was added to chitosan solution to prepare final concentrations of thyroxine 0.1μg/mL, 0.5μg/mL and 1.0μg/mL. Then non-sintered HAp at a concentration of 1% w/v was added in already prepared solution. Finally, powdered carboxymethyl cellulose (2% w/v) was added while stirring. The mixture was frozen at -40 °C for 48 h and finally lyophilized in a freeze dryer (Christ®, Model: Alpha 1-2 LD plus) for 36 h to obtain hydrogels of 8.5 cm diameter (Fig 1). The concentration of each material in the respective hydrogels and their codes are given in table 1. Table 1: Formulation and codes of thyroxine-loaded chitosan/carboxymethyl cellulose/ hydroxyapatite hydrogels
CODES
Control
TX-L
TX-M
TX-H
Thyroxine
-
0.1μg/mL
0.5μg/mL
1.0μg/mL
Chitosan
2% w/v
2% w/v
2% w/v
2% w/v
Carboxymethyl
2% w/v
2% w/v
2% w/v
2% w/v
1%w/v
1%w/v
1%w/v
1%w/v
cellulose Hydroxyapatite
2.3- Fourier Transform Infrared Spectroscopy IR spectra were taken using Fourier Transform Infrared Spectroscopy (Thermo Nicolet 6700P, USA). The spectra were recorded at room temperature using smart ATR (Attenuated Total Reflectance) mode with carbon background and nitrogen purging, with resolution of 8 cm-1, 128 scan numbers and range of 4000–650 cm-1.
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2.4- Scanning electron microscopy (SEM) SEM images of the hydrogel cross-section were taken using Scanning Electron Microscope (VEGA3 TESCAN). An accelerating voltage of 10 kV was applied and samples were sputter coated with gold. The average pore size was calculated by using image-processing software (Image J). 2.5- Drug Release Studies The experiment was designed for evaluating thyroxine release from CS gel membranes. The method was adopted from a reported study with some modifications [33]. Membranes were cut in small pieces weighing 12 mg ± 2 each. Thyroxine stock solution (1 ug/ mL) was prepared in PBS at room temperature. Each membrane piece was placed in separate sterile vials containing 5 mL of thyroxine solution. For release studies, time points were selected 0, 1, 2, 6 and 7 days. Each membrane was placed in separate sterile vials containing 5 mL PBS. At each selected time point, the membrane were removed and placed in fresh PBS media (5ml). The release media was used to measure thyroxine release using UV–Vis spectrophotometer at 227 nm. The data was plotted as cumulative release (%) as a function of time.
2.6- Swelling studies To study the swelling behavior of hydrogels, 10 ± 1 mg hydrogel sample from each group was taken and placed in PBS (Phosphate Buffer Saline, pH 7.4) at 37°C. Hydrogels were taken out of PBS, placed on filter/tissue paper for 1 sec to remove surface water and then were weighed at 1/2h, 1h, 3h, 12h and 24h after first incubation. The swelling ratio was then calculated by using the following formula (eq. 1) where W represents the weight of hydrogel at particular interval and WD denotes the initial weight of dry hydrogel: 𝑠𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑟𝑎𝑡𝑖𝑜 = 100 × W − WD⁄𝑊𝐷 2.8- Cell adhesion and proliferation
5
eq. 1
MC3T3-E1 cells were maintained in complete culture medium containing MEM-α, supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin in T25 flasks. 1mL Trypsin-EDTA solution was used to detach the cells. During sub-culturing, cells were washed with sterile PBS to ensure a total removal of medium and cell debris. Cells were grown under standard cell culture conditions (37oC and a humidified atmosphere of 5% CO2). To see the effect of our hydrogels, cells were subsequently seeded in a 12 well plate, containing hydrogels control, TX-L, TX-M and TX-H. One well was left blank as a control, with same number of cells but no hydrogel. Total 1.9x104 MC3T3-E1 cells were seeded in each well (5000 cells/cm2) and incubated for 72 hours at 37oC with 5% CO2. Cells were observed under VWR inverted fluorescence microscope. 2.9- Chick chorioallantoic membrane (CAM) assay Fertilized chicken eggs (Gallus domesticus) were purchased from Big Bird (Lahore, Pakistan) on day 0 and kept in egg incubator (R-COM Suro20) at 37oC, 40% humidity. On day 7, a small window (1 cm2) from shell was made on each egg. Small pieces (0.5 cm2) of each sample (hydrogels) were implanted (one sample per egg) through the window, then were covered with parafilm and sealed with adhesive tape. The eggs were further incubated for another 7 days till chick becomes 14 days old. At day 14, sealing tapes and parafilm were taken off and images of hydrogels inside the eggs were taken. Finally, hydrogels were retrieved and fixed in formaldehyde solution (4%). Images of these fixed hydrogels were again taken to evaluate the penetration of blood vessels inside the hydrogel [34-37]. 2.10- Ex-ovo Cavity Filling Demonstration A 2 cm × 2 cm piece of hydrogel was cut and placed in the petri dish. Approximately 2–4 mL of distilled water was also added onto the hydrogel. The hydrogel piece was crushed in the water and sucked into the syringe without needle. A small piece of beef was taken and a cavity (1.5 cm deep) was formed in the meat. The cavity was filled in by crushed hydrogel via syringe and images of each step were taken accordingly. 3- Results 3.1- Preparation of hydrogels
6
Hydrogels were prepared by simple mixing of all materials in a sequence. First, chitosan solution was prepared and thyroxine was dissolved in it. Then, the chitosan/thyroxine solution was added in another separately prepared chitosan solution, in different amounts, to prepare solutions with different concentrations of thyroxine. To these solutions, hydroxyapatite was added, which was followed by addition of carboxymethyl cellulose. It transformed the solution into white, gel-like mixture that was further added to the petri-dish for lyophilization (Fig 1).
Fig. 1: Overview of the thyroxine-loaded chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels preparation– testing including cytotoxicity and CAM assay.
Fig. 2 showed that hydrogels could be easily bended, folded, rolled and stretched. It has been observed that for wet hydrogels, with the help of a tweezer, physical bending, folding, rolling and stretching were observed. As depicted in Fig 2 the physical strength of the hydrogel, after being wet, remained intact and could have easily been transformed into any shape or form. Also, the wet hydrogel was stretchable upon application of mild physical force.
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Fig 2: Physical appearance of the hydrogel when (a) soaked in a few drops of water, (b) bended with a tweezer, (c) fully bended, (d), (e) rolled-over and (f) stretched.
3.2- Structural analysis Fig 3 depicts FTIR spectra of composite hydrogels (control, TX-L, TX-M and TX-H) and starting materials, chitosan, carboxymethyl cellulose, thyroxine and hydroxyapatite. The presence of each constituent material was confirmed by appearance of characteristic peaks for different functional groups. The broad peak at 3400–3200 cm-1 was attributed to O-H and N-H stretching vibration of chitosan, which was also visible in FTIR spectra of hydrogels. Similarly, the characteristic peak of chitosan for amide I appeared at 1655 cm-1 [38, 39]. Carboxymethyl cellulose expressed characteristic peaks at 1423 cm-1 for methyl group (methyl stretch), this was also present in all hydrogels at the same wavenumber. The characteristic peak for carbonyl group of thyroxine appeared between 1630 cm-1 and 1650 cm-1.
8
Fig 3: FTIR spectra of hydrogels (a) control, (b) TX-L, (c) TX-M and (d) TX-H and starting materials, (e) thyroxine, (f) hydroxyapatite, (g) chitosan and (h) carboxymethyl cellulose.
3.3- Morphology and physical appearance of the hydrogels All hydrogels exhibited minimal shrinkage after freeze drying as shown in Fig 4A. Microstructure revealed large pores and spaces in all lyophilized hydrogels with uniform distribution. The average pore diameters for control, TX-L, TX-M and TX-H was found to be 98.19μm + 47.74μm, 96.63μm + 54.28μm, 90.59μm + 42.2μm and 128.97μm + 68.21μm (average pore diameter of 20 randomly selected pores was taken) respectively (Fig 4B). The cross-sectioned images also show interconnected pore structure despite of large pores in the hydrogels.
9
Fig 4A: The physical appearance of hydrogels after lyophilization (diameter=8.5 cm) (a) control, (b) TX-L, (c) TXM and (d) TX-H. SEM images of hydrogels showing pore morphology at scale 250 μm and 50 μm.
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Fig 4B: Bar chart showing average pore size of (a) control, (b) TX-L, (c) TX-M and (d) TX-H hydrogels.
3.4- In – vitro Drug Release Studies Time dependent drug release from thyroxine loaded membranes (TX-L, TX-M & TX-H) were monitored in PBS solution by UV-visible spectroscopy at 227 nm. In Fig. 5 drug release profile is shown. On day 1, TX-L, TX-M and TX-H exhibited 18 %, 25 % and 45 % release of thyroxin respectively. On day 2, materials revealed 48 % (TX-L), 65 % (TX-M) and 78 % (TX-H) release of thyroxin. On day 6, 78 % (TX-L), 86 % (TX-M) and 89 % (TX-H) thyroxin release was observed. Over 7 days period, total cumulative percentage release was 89%, 92% and 97% from TX-L, TX-M and TX-H respectively.
11
Figure 5: Cumulative percentage drug release profile of thyroxine with different concentrations (TX-L, TX-M & TX-H) in PBS solution.
3.4- Swelling capacity Swelling capacity of control, TX-L, TX-M and TX-H reached to their respective maxima after 0.5 h of incubation in PBS solution. Swelling ratios observed (Fig 6) for control, TX-L, TX-M and TX-H were 2856.5%, 2181.9%, 2413.2% and 2473.7% respectively.
12
Fig 6: Bar chart showing swelling ratios of individual hydrogels after 0.5 h.
3.6-Biocompatibility of the hydrogel For the potential use of hydrogels in tissue engineering, the structure and chemical composition of that hydrogel must ensure a normal growth and morphology of cells [15, 40], with no toxic effect on the cellular machinery and biological pathways. We aimed to observe any deleterious effect of synthesized hydrogels on the morphology and growth of MC3T3-E1 cells. In our experiment, MC3T3-E1 cells exhibited a normal morphology and growth in the presence of hydrogels, (a) control, (b) TX-L, (c) TX-M and (d) TX-H (Fig 7). Cells were found viable, demonstrating these hydrogels provide a non-toxic environment in which osteoblasts can grow and proliferate.
13
Fig 7: Inverted microscope images of the pre-osteoblast cells (MC3T3) at day 3 for (a) negative control, (b) positive control/ control, (b) TX-L, (c) TX-M and (d) TX-H
3.7- Angiogenic potential by using Chorioallantoic membrane Assay (CAM) The CAM assay (Fig 8A) displayed incorporation of all hydrogels in CAM. Blood vessels were seen grown around each hydrogel. Minimal growth of blood vessels was observed in control, whereas blood vessels seemed to have penetrated all other hydrogels (loaded with thyroxine). The infiltration pattern of blood vessels was also confirmed by hydrogels removed from the eggs (explants). Highest number of blood vessel was found in TX-L after blind counting (n=3), followed by TX-M. Among thyroxine loaded hydrogels, least angiogenesis was observed in samples containing 1μg/mL thyroxine (TX-H). Paired t-test also confirmed the significantly high penetration and ingrowth of blood vessels in TX-L compared to control (Fig 8B and 8C).
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Fig 8: (A) CAM assay images of hydrogels in-vivo and ex-plant; average blood vessel count (blinded) of each hydrogel (B) in-vivo count of blood vessels and (C) number of blood vessels in ex-plant scaffolds.
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3.8- Ex-ovo testing of the thyroxin loaded hydrogel In this applied experiment, the hydrogel showed adhesive behavior, both while filling (Fig 9c) and after getting filled (Fig 9d) in the cavity. When the piece of meat was hanged vertically, the gel did not flow out (Fig 9e) even after a few minutes.
Fig 9: (a) close-up view of cavity dug in a piece of meat, (b) syringe filled with diluted hydrogel, (c) filling of cavity with hydrogel via syringe, (d) cavity filled with diluted hydrogel as placed on a horizontal surface and (e) retention of diluted hydrogel in vertical position.
Discussion A number of research articles have reported the loading of various growth factors on scaffolds made from different combinations of polymers for periodontal regeneration, e.g. porous chitosan/coral composites have been loaded with platelet-derived growth factors [41], polylactic acid-poly glycolic acid copolymer and gelatin sponge with bone morphogenetic protein [42] and collagen gel with fibroblast growth factor 2 [43]. These combinations were either used to support or accelerate the process of regeneration. The majority of commercially available membranes for periodontal regeneration are either based on polyesters (e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(ɛ-caprolactone) (PCL), and their copolymers) or are collagen based [44]. The synthetic polymers owing to their high costs and collagen-based membranes due to their ambiguity 16
in source of derivation, further leading to mainly religious concerns would not be administered in majority of patients. Nevertheless, none of these developed materials intended to induce angiogenesis for periodontal regeneration. We prepared hydrogels by directly mixing all starting materials, chitosan, carboxymethyl cellulose, thyroxine and hydroxyapatite and chose lyophilization as the tool of fabrication. Thyroxine had been made a part of the hydrogels in different concentrations, owing to its potential to induce angiogenesis by activating vascular endothelial growth factor (VEGF) [15, 19, 28]. The starting polymeric materials are well-known for their biocompatibility, along with hydroxyapatite which is a naturally and most abundantly found mineral in hard tissues (tooth and bone) in mammals (including humans). The objective of this research was to develop hydrogels, having capacity to hold thyroxine to induce angiogenesis for potential use in periodontal regeneration. Blood vessels formation is one of the pre-requisites to repair and regenerate most tissues, periodontal cavity being no exception. Chitosan alone, when freeze dried, cannot produce scaffolds with good mechanical properties [45]. Carboxymethyl cellulose is a Food and Drug Administration approved material, termed as GRAS (Generally Recognized as Safe) by FDA [46]. Addition of carboxymethyl cellulose helped improving the physical strength of the hydrogels when freeze dried. Also, the nearly uniform distribution of hydroxyapatite within hydrogel can be attributed to the sticky nature of carboxymethyl cellulose that kept particles stranded and did not allow them to settle when poured in the petri dishes. FTIR spectra provided us with the confirmation of the presence of individual starting materials in hydrogels control, TX-L, TX-M and TX-H exhibiting characteristic peaks of all the functional groups present. Appearance of hydroxyapatite peaks in all samples also indicated the presence of hydroxyapatite on the surface, in addition to distribution within hydrogels (as indicated by SEM, Fig 4a). FTIR also portrayed absence of any chemical interaction among materials as well as with thyroxine. Large pores were observed in the hydrogels after lyophilization, which can be attributed to the high-water holding capacity of carboxymethyl cellulose and chitosan. Thus, when the mixture was frozen, large water crystals would have formed, which when further lyophilized, left bigger spaces
17
as pores. These large pores were responsible for immediate and very high swelling ratios when 10mg+1mg of each scaffold was immersed in the PBS solution. Angiogenesis is very critical for proper and fast tissue regeneration, VEGF is very well known for stimulating angiogenesis [47], the onsite delivery of VEGF strategy could not sustain in market due to high cost and low stability. Thyroxine is one of the very promising alternative to VEGF, the sustained delivery of thyroxine to wounded area or the tissue undergoing regeneration will be helpful for fast recovery and tissue repair respectively. In current research we have observed that thyroxine was releasing from biomaterials for 7 days, on day 1 around 45% and till day 6 around 85% drug was released. This shows that these are promising materials for tissue repair and regeneration. CAM assay was used to describe the angiogenic potential of the prepared hydrogels. Minimal vessels were noticed around and inside the control scaffold, both in-vivo and ex-plant. Though blood vessels can be seen near control, little penetration had been observed in explant (Fig 9a). Maximum blood vessels were grown in and around TX-L, which was further proved by significant values for paired t-test, when compared with control both in-vivo and ex-plant (Fig 9b and c). The trend of blood vessels ingrowth decreased as the thyroxine concentration increased (TX-L > TXM > TX-H). This result helped us not only to quantify and select the best angiogenic material but also proved that thyroxine encourages the growth and penetration of endothelial cells in chitosan/carboxymethyl cellulose hydrogel at low concentrations. It is shown by ex-vivo cavity filling experiment that developed hydrogel can be administered to a cavity with the help of a syringe. It is proposed that synthesized hydrogel can be used to fill periodontal cavity either by folding around a slightly soaked piece or directly administering diluted gel in the cavity. Adhesion of diluted gel in the meat cavity in ex-vivo experiment suggested that it can remain at the site of administration for an extended period, easily. Conclusion In current research, thyroxine was added to the chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels as angiogenic agent in different concentrations. The homogeneity of mixture was achieved by adding carboxymethyl cellulose powder in chitosan/HAp/thyroxine suspension. The physical characterization confirmed the presence and homogeneous distribution of all materials. 18
Hydrogels possessed interconnected porosity which resulted in rapid solution absorption and high swelling ratio. It was concluded by CAM assay that the lower concentration of thyroxine (0.1 µg) in hydrogels promoted greater angiogenesis than higher concentrations. The formation and infiltration of new blood vessels were observed in thyroxin containing ex-planted scaffolds. These studies proved that these materials are excellent candidates for periodontal regeneration.
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Acknowledgement We acknowledge Higher Education Commision project # Project# HEC-TDF-094, Pakistan Science Foundation for funding this research project# PSF-NSF/Med/P-COMSATS (01) and Ministry of Science and Technology Pakistan for financial support. We would like to acknowledge Samreen Ahtzaz and Abdur Raheem Aleem for their support and cooperation during this research work. We would also like to thank Dr Farasat Iqbal and Faisal Manzoor for their help in SEM.
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S0141-8130(19)34614-8 https://doi.org/10.1016/j.ijbiomac.2019.10.043 BIOMAC 13492
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
19 June 2019 25 September 2019 3 October 2019
Please cite this article as: M. Hamza Malik, L. Shahzadi, R. Batool, S. Zaman Safi, A. Samad Khan, A. Farooq Khan, A. Anwar Chaudhry, I. Ur Rehman, M. Yar, Thyroxine-loaded chitosan/carboxymethyl cellulose/ hydroxyapatite hydrogels enhance angiogenesis in ex-ovo experiments, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.043
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Thyroxine-loaded chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels enhance angiogenesis in ex-ovo experiments Muhammad Hamza Malika, Lubna Shahzadia, Razia Batoola, Sher Zaman Safia, Abdul Samad Khana,c, Ather Farooq Khana, Aqif Anwar Chaudhrya, Ihtesham Ur Rehmana,b, Muhammad Yara,* a
Interdisciplinary Research Center in Biomedical Materials, COMSATS University Islamabad, Lahore Campus,54000, Pakistan b Engineering Department, Lancaster University, Lancaster, UK c Department of Restorative Dental Sciences, College of Dentistry, University of Dammam,31441, Saudi Arabia
*Correspondence Address: a
Interdisciplinary Research Center in Biomedical Materials, COMSATS University Islamabad, Lahore Campus,54000, Pakistan; Tel: 0092 42 001007 ext 829; Fax: 0092 42 35321090; E-mail: [email protected] (M. Yar)
Abstract Angiogenesis is one of the most important processes in repair and regeneration of many tissues and organs. Blood vessel formation also play a major role in repair of dental tissue(s) after ailments like
periodontitis.
Here
we
report
the
preparation
of
chitosan/carboxymethyl
cellulose/hydroxyapatite based hydrogels, loaded with variable concentrations of thyroxin i.e., 0.1 μg/ml, 0.5 μg/ml and 1 μg/ml. Scanning electron microcopy images (SEM) showed all hydrogels
were found to be porous and solution absorption study exhibited high swelling potential in aqueous media. FTIR spectra confirmed that the used materials did not change their chemical identity in synthesized hydrogels. The synthesized hydrogels demonstrated good bending, folding, rolling and stretching abilities. The hydrogels were tested in chick chorioallantoic membrane (CAM) assay to investigate their angiogenic potential. Hydrogel containing 0.1 μg/ml of thyroxine showed maximum neovascularization. For cytotoxicity analyses, preosteoblast cells (MC3T3-E1) were seeded on these hydrogels and materials were found to be non-toxic. These hydrogels with proangiogenic activity possess great potential to be used for periodontal regeneration. Key words: Tissue engineering, periodontal regeneration, thyroxine, angiogenesis, chitosan, carboxymethyl cellulose, hydroxyapatite, injectable gels Introduction 1
Tissue engineering has gained popularity over years as a potential solution in the field of regenerative medicine; whereby natural, semisynthetic and synthetic materials having medical response are implanted to mimic the natural response of the failed tissue or organ[1, 2]. Angiogenesis has been one of the determining factors for the regeneration of different tissues and organs in human body. It is a vital process in wound healing, as newly formed blood vessels helps to form the granulation tissue and provide oxygen and nutrition to the injured area [3]. It is also one of the essential requirements for hard tissue regeneration. A number of research reports have expressed the importance of angiogenesis in healing and repair of fractured bone [4], e.g. Schmidt et. al., found that blood vessel formation occurs before new bone formation in rabbits. [5]. Destruction of periodontal tissue is termed as periodontal disease and characterized by the necrosis of gingiva, alveolar bone, periodontal ligament and cementum [6]. According to Center for Disease Control and Prevention, 47.8% of the adults aged 30 or above were affected by some form of periodontal disease in USA, 8.7% having mild, 30.0% having moderate, and 8.5% with severe form of periodontitis [7]. The prevalence of periodontitis has been found to be 64% in older adults, aged 65 years and older in US [7]. Generally, scaling and curettage, medications including mouthwashes, antibiotics and surgical interventions have been employed to treat periodontal diseases [8], however little has been achieved to completely reverse the disease progression and regenerate the damaged periodontal tissue. Periodontal disease has been related to other systemic diseases and disorders as well, like adverse events in pregnancy [9], oral cancer [10] and diabetes [11], which shows the need to form new interventions to permanently treat the disease. Periodontium is richly supplied with blood vessels and induction of angiogenesis is necessary in order to supply nutrients and oxygen to the grafted tissue engineered construct for better repair [12]. Another study demonstrated that enamel matrix derivative induces angiogenesis during periodontal wound healing [13]. In addition to periodontium, angiogenesis has been found to be essential in the regeneration of pulp [14, 15], which results in the production of dentin-like tissue along the dentinal wall by continuous layer of odontoblast –like cells [16]. Growth factors that are generally involved in angiogenesis are found to be up-regulated in periodontal repair process. The fibroblast growth factor (bFGF) promotes the cell growth of periodontal ligament and up-regulates the laminin mRNA level, which further promotes the process of angiogenesis [15, 17].
2
Thyroxine is one of the vital hormones with various physiological functions within human body. One of the functions, associated with thyroxine is its ability to induce angiogenesis by several mechanisms [18]. Mainly, thyroxine causes the transcription of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), the two main growth factors responsible for blood vessel growth by activating integrin αvβ3 [19]. A number of polymeric biomaterials such as chitosan, polycaprolactone and hydroxyethyl cellulose have been shown to possess potential for periodontal treatment by helping to regenerate the tissue and/or delivering drugs [20]. Chitosan is a natural copolymer comprising of glucosamine and N-acetylglucosamine, generally derived from crustacean shells by partial deacetylation of chitin [6] and it has been used alone or with other polymers [21-23] as a biomaterial for its attributes of being biocompatible, biodegradable and bacteriostatic [18, 24, 25]. Many research groups have shown potential of chitosan for periodontal treatment and regeneration [26]. Hydroxyapatite (HA), a naturally occurring bioactive material, has been incorporated in chitosan by wet chemical method [27, 28] or lyophilization [29]. Carboxymethyl cellulose is another polymer with well-known use in food and pharmaceutical industry. Due to its slow degradability and biocompatibility, it has been used for hard tissue regeneration along with chitosan and HAp [30, 31]. Hydroxyapatite is a naturally occurring material, responsible for the hardness of bone, enamel and dentine [31, 32]. Since there is absolute need for biocompatible membranes which can support angiogenesis and at the same time help tissue regeneration. In current research, thyroxine loaded biocompatible proangiogenic hydrogels have been developed from chitosan, carboxymethyl cellulose and hydroxyapatite. The synthesized membranes showed good foldability and stretching properties and upon supplying slight liquid these membranes can be converted into injectable hydrogels. CAM assay showed the angiogenic activity of the synthesized materials and variable amounts of thyroxin were tested to find the suitable concentration required for efficient angiogenesis. 2- Materials and Methods 2.1- Materials All chemicals used in this study were analytical grade. Chitosan (CS) was purchased from Mian Scientific Company (Lahore, Pakistan) and further purified as we described earlier [32] (Mw: 3
144,105.g/mol., degree of deacetylation (DD): 83%, intrinsic viscosity: 30.85 ml/g). HAp was also synthesized in our laboratory [33]. Glacial acetic acid (BDH Laboratory Supplies UK) was of analytical grade and was used without further purification. Thyroxine was provided by GlaxoSmithKline Pakistan. Mouse pre-osteoblast cell line MC3T3-E1 sub-clone 14 was purchased from ATCC cell bank (USA). 2.2- Preparation of freeze-dried hydrogels Chitosan (2% w/v) was dissolved in 1% acetic acid solution and the thyroxine solution was added to chitosan solution to prepare final concentrations of thyroxine 0.1μg/mL, 0.5μg/mL and 1.0μg/mL. Then non-sintered HAp at a concentration of 1% w/v was added in already prepared solution. Finally, powdered carboxymethyl cellulose (2% w/v) was added while stirring. The mixture was frozen at -40 °C for 48 h and finally lyophilized in a freeze dryer (Christ®, Model: Alpha 1-2 LD plus) for 36 h to obtain hydrogels of 8.5 cm diameter (Fig 1). The concentration of each material in the respective hydrogels and their codes are given in table 1. Table 1: Formulation and codes of thyroxine-loaded chitosan/carboxymethyl cellulose/ hydroxyapatite hydrogels
CODES
Control
TX-L
TX-M
TX-H
Thyroxine
-
0.1μg/mL
0.5μg/mL
1.0μg/mL
Chitosan
2% w/v
2% w/v
2% w/v
2% w/v
Carboxymethyl
2% w/v
2% w/v
2% w/v
2% w/v
1%w/v
1%w/v
1%w/v
1%w/v
cellulose Hydroxyapatite
2.3- Fourier Transform Infrared Spectroscopy IR spectra were taken using Fourier Transform Infrared Spectroscopy (Thermo Nicolet 6700P, USA). The spectra were recorded at room temperature using smart ATR (Attenuated Total Reflectance) mode with carbon background and nitrogen purging, with resolution of 8 cm-1, 128 scan numbers and range of 4000–650 cm-1.
4
2.4- Scanning electron microscopy (SEM) SEM images of the hydrogel cross-section were taken using Scanning Electron Microscope (VEGA3 TESCAN). An accelerating voltage of 10 kV was applied and samples were sputter coated with gold. The average pore size was calculated by using image-processing software (Image J). 2.5- Drug Release Studies The experiment was designed for evaluating thyroxine release from CS gel membranes. The method was adopted from a reported study with some modifications [33]. Membranes were cut in small pieces weighing 12 mg ± 2 each. Thyroxine stock solution (1 ug/ mL) was prepared in PBS at room temperature. Each membrane piece was placed in separate sterile vials containing 5 mL of thyroxine solution. For release studies, time points were selected 0, 1, 2, 6 and 7 days. Each membrane was placed in separate sterile vials containing 5 mL PBS. At each selected time point, the membrane were removed and placed in fresh PBS media (5ml). The release media was used to measure thyroxine release using UV–Vis spectrophotometer at 227 nm. The data was plotted as cumulative release (%) as a function of time.
2.6- Swelling studies To study the swelling behavior of hydrogels, 10 ± 1 mg hydrogel sample from each group was taken and placed in PBS (Phosphate Buffer Saline, pH 7.4) at 37°C. Hydrogels were taken out of PBS, placed on filter/tissue paper for 1 sec to remove surface water and then were weighed at 1/2h, 1h, 3h, 12h and 24h after first incubation. The swelling ratio was then calculated by using the following formula (eq. 1) where W represents the weight of hydrogel at particular interval and WD denotes the initial weight of dry hydrogel: 𝑠𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑟𝑎𝑡𝑖𝑜 = 100 × W − WD⁄𝑊𝐷 2.8- Cell adhesion and proliferation
5
eq. 1
MC3T3-E1 cells were maintained in complete culture medium containing MEM-α, supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin in T25 flasks. 1mL Trypsin-EDTA solution was used to detach the cells. During sub-culturing, cells were washed with sterile PBS to ensure a total removal of medium and cell debris. Cells were grown under standard cell culture conditions (37oC and a humidified atmosphere of 5% CO2). To see the effect of our hydrogels, cells were subsequently seeded in a 12 well plate, containing hydrogels control, TX-L, TX-M and TX-H. One well was left blank as a control, with same number of cells but no hydrogel. Total 1.9x104 MC3T3-E1 cells were seeded in each well (5000 cells/cm2) and incubated for 72 hours at 37oC with 5% CO2. Cells were observed under VWR inverted fluorescence microscope. 2.9- Chick chorioallantoic membrane (CAM) assay Fertilized chicken eggs (Gallus domesticus) were purchased from Big Bird (Lahore, Pakistan) on day 0 and kept in egg incubator (R-COM Suro20) at 37oC, 40% humidity. On day 7, a small window (1 cm2) from shell was made on each egg. Small pieces (0.5 cm2) of each sample (hydrogels) were implanted (one sample per egg) through the window, then were covered with parafilm and sealed with adhesive tape. The eggs were further incubated for another 7 days till chick becomes 14 days old. At day 14, sealing tapes and parafilm were taken off and images of hydrogels inside the eggs were taken. Finally, hydrogels were retrieved and fixed in formaldehyde solution (4%). Images of these fixed hydrogels were again taken to evaluate the penetration of blood vessels inside the hydrogel [34-37]. 2.10- Ex-ovo Cavity Filling Demonstration A 2 cm × 2 cm piece of hydrogel was cut and placed in the petri dish. Approximately 2–4 mL of distilled water was also added onto the hydrogel. The hydrogel piece was crushed in the water and sucked into the syringe without needle. A small piece of beef was taken and a cavity (1.5 cm deep) was formed in the meat. The cavity was filled in by crushed hydrogel via syringe and images of each step were taken accordingly. 3- Results 3.1- Preparation of hydrogels
6
Hydrogels were prepared by simple mixing of all materials in a sequence. First, chitosan solution was prepared and thyroxine was dissolved in it. Then, the chitosan/thyroxine solution was added in another separately prepared chitosan solution, in different amounts, to prepare solutions with different concentrations of thyroxine. To these solutions, hydroxyapatite was added, which was followed by addition of carboxymethyl cellulose. It transformed the solution into white, gel-like mixture that was further added to the petri-dish for lyophilization (Fig 1).
Fig. 1: Overview of the thyroxine-loaded chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels preparation– testing including cytotoxicity and CAM assay.
Fig. 2 showed that hydrogels could be easily bended, folded, rolled and stretched. It has been observed that for wet hydrogels, with the help of a tweezer, physical bending, folding, rolling and stretching were observed. As depicted in Fig 2 the physical strength of the hydrogel, after being wet, remained intact and could have easily been transformed into any shape or form. Also, the wet hydrogel was stretchable upon application of mild physical force.
7
Fig 2: Physical appearance of the hydrogel when (a) soaked in a few drops of water, (b) bended with a tweezer, (c) fully bended, (d), (e) rolled-over and (f) stretched.
3.2- Structural analysis Fig 3 depicts FTIR spectra of composite hydrogels (control, TX-L, TX-M and TX-H) and starting materials, chitosan, carboxymethyl cellulose, thyroxine and hydroxyapatite. The presence of each constituent material was confirmed by appearance of characteristic peaks for different functional groups. The broad peak at 3400–3200 cm-1 was attributed to O-H and N-H stretching vibration of chitosan, which was also visible in FTIR spectra of hydrogels. Similarly, the characteristic peak of chitosan for amide I appeared at 1655 cm-1 [38, 39]. Carboxymethyl cellulose expressed characteristic peaks at 1423 cm-1 for methyl group (methyl stretch), this was also present in all hydrogels at the same wavenumber. The characteristic peak for carbonyl group of thyroxine appeared between 1630 cm-1 and 1650 cm-1.
8
Fig 3: FTIR spectra of hydrogels (a) control, (b) TX-L, (c) TX-M and (d) TX-H and starting materials, (e) thyroxine, (f) hydroxyapatite, (g) chitosan and (h) carboxymethyl cellulose.
3.3- Morphology and physical appearance of the hydrogels All hydrogels exhibited minimal shrinkage after freeze drying as shown in Fig 4A. Microstructure revealed large pores and spaces in all lyophilized hydrogels with uniform distribution. The average pore diameters for control, TX-L, TX-M and TX-H was found to be 98.19μm + 47.74μm, 96.63μm + 54.28μm, 90.59μm + 42.2μm and 128.97μm + 68.21μm (average pore diameter of 20 randomly selected pores was taken) respectively (Fig 4B). The cross-sectioned images also show interconnected pore structure despite of large pores in the hydrogels.
9
Fig 4A: The physical appearance of hydrogels after lyophilization (diameter=8.5 cm) (a) control, (b) TX-L, (c) TXM and (d) TX-H. SEM images of hydrogels showing pore morphology at scale 250 μm and 50 μm.
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Fig 4B: Bar chart showing average pore size of (a) control, (b) TX-L, (c) TX-M and (d) TX-H hydrogels.
3.4- In – vitro Drug Release Studies Time dependent drug release from thyroxine loaded membranes (TX-L, TX-M & TX-H) were monitored in PBS solution by UV-visible spectroscopy at 227 nm. In Fig. 5 drug release profile is shown. On day 1, TX-L, TX-M and TX-H exhibited 18 %, 25 % and 45 % release of thyroxin respectively. On day 2, materials revealed 48 % (TX-L), 65 % (TX-M) and 78 % (TX-H) release of thyroxin. On day 6, 78 % (TX-L), 86 % (TX-M) and 89 % (TX-H) thyroxin release was observed. Over 7 days period, total cumulative percentage release was 89%, 92% and 97% from TX-L, TX-M and TX-H respectively.
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Figure 5: Cumulative percentage drug release profile of thyroxine with different concentrations (TX-L, TX-M & TX-H) in PBS solution.
3.4- Swelling capacity Swelling capacity of control, TX-L, TX-M and TX-H reached to their respective maxima after 0.5 h of incubation in PBS solution. Swelling ratios observed (Fig 6) for control, TX-L, TX-M and TX-H were 2856.5%, 2181.9%, 2413.2% and 2473.7% respectively.
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Fig 6: Bar chart showing swelling ratios of individual hydrogels after 0.5 h.
3.6-Biocompatibility of the hydrogel For the potential use of hydrogels in tissue engineering, the structure and chemical composition of that hydrogel must ensure a normal growth and morphology of cells [15, 40], with no toxic effect on the cellular machinery and biological pathways. We aimed to observe any deleterious effect of synthesized hydrogels on the morphology and growth of MC3T3-E1 cells. In our experiment, MC3T3-E1 cells exhibited a normal morphology and growth in the presence of hydrogels, (a) control, (b) TX-L, (c) TX-M and (d) TX-H (Fig 7). Cells were found viable, demonstrating these hydrogels provide a non-toxic environment in which osteoblasts can grow and proliferate.
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Fig 7: Inverted microscope images of the pre-osteoblast cells (MC3T3) at day 3 for (a) negative control, (b) positive control/ control, (b) TX-L, (c) TX-M and (d) TX-H
3.7- Angiogenic potential by using Chorioallantoic membrane Assay (CAM) The CAM assay (Fig 8A) displayed incorporation of all hydrogels in CAM. Blood vessels were seen grown around each hydrogel. Minimal growth of blood vessels was observed in control, whereas blood vessels seemed to have penetrated all other hydrogels (loaded with thyroxine). The infiltration pattern of blood vessels was also confirmed by hydrogels removed from the eggs (explants). Highest number of blood vessel was found in TX-L after blind counting (n=3), followed by TX-M. Among thyroxine loaded hydrogels, least angiogenesis was observed in samples containing 1μg/mL thyroxine (TX-H). Paired t-test also confirmed the significantly high penetration and ingrowth of blood vessels in TX-L compared to control (Fig 8B and 8C).
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Fig 8: (A) CAM assay images of hydrogels in-vivo and ex-plant; average blood vessel count (blinded) of each hydrogel (B) in-vivo count of blood vessels and (C) number of blood vessels in ex-plant scaffolds.
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3.8- Ex-ovo testing of the thyroxin loaded hydrogel In this applied experiment, the hydrogel showed adhesive behavior, both while filling (Fig 9c) and after getting filled (Fig 9d) in the cavity. When the piece of meat was hanged vertically, the gel did not flow out (Fig 9e) even after a few minutes.
Fig 9: (a) close-up view of cavity dug in a piece of meat, (b) syringe filled with diluted hydrogel, (c) filling of cavity with hydrogel via syringe, (d) cavity filled with diluted hydrogel as placed on a horizontal surface and (e) retention of diluted hydrogel in vertical position.
Discussion A number of research articles have reported the loading of various growth factors on scaffolds made from different combinations of polymers for periodontal regeneration, e.g. porous chitosan/coral composites have been loaded with platelet-derived growth factors [41], polylactic acid-poly glycolic acid copolymer and gelatin sponge with bone morphogenetic protein [42] and collagen gel with fibroblast growth factor 2 [43]. These combinations were either used to support or accelerate the process of regeneration. The majority of commercially available membranes for periodontal regeneration are either based on polyesters (e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(ɛ-caprolactone) (PCL), and their copolymers) or are collagen based [44]. The synthetic polymers owing to their high costs and collagen-based membranes due to their ambiguity 16
in source of derivation, further leading to mainly religious concerns would not be administered in majority of patients. Nevertheless, none of these developed materials intended to induce angiogenesis for periodontal regeneration. We prepared hydrogels by directly mixing all starting materials, chitosan, carboxymethyl cellulose, thyroxine and hydroxyapatite and chose lyophilization as the tool of fabrication. Thyroxine had been made a part of the hydrogels in different concentrations, owing to its potential to induce angiogenesis by activating vascular endothelial growth factor (VEGF) [15, 19, 28]. The starting polymeric materials are well-known for their biocompatibility, along with hydroxyapatite which is a naturally and most abundantly found mineral in hard tissues (tooth and bone) in mammals (including humans). The objective of this research was to develop hydrogels, having capacity to hold thyroxine to induce angiogenesis for potential use in periodontal regeneration. Blood vessels formation is one of the pre-requisites to repair and regenerate most tissues, periodontal cavity being no exception. Chitosan alone, when freeze dried, cannot produce scaffolds with good mechanical properties [45]. Carboxymethyl cellulose is a Food and Drug Administration approved material, termed as GRAS (Generally Recognized as Safe) by FDA [46]. Addition of carboxymethyl cellulose helped improving the physical strength of the hydrogels when freeze dried. Also, the nearly uniform distribution of hydroxyapatite within hydrogel can be attributed to the sticky nature of carboxymethyl cellulose that kept particles stranded and did not allow them to settle when poured in the petri dishes. FTIR spectra provided us with the confirmation of the presence of individual starting materials in hydrogels control, TX-L, TX-M and TX-H exhibiting characteristic peaks of all the functional groups present. Appearance of hydroxyapatite peaks in all samples also indicated the presence of hydroxyapatite on the surface, in addition to distribution within hydrogels (as indicated by SEM, Fig 4a). FTIR also portrayed absence of any chemical interaction among materials as well as with thyroxine. Large pores were observed in the hydrogels after lyophilization, which can be attributed to the high-water holding capacity of carboxymethyl cellulose and chitosan. Thus, when the mixture was frozen, large water crystals would have formed, which when further lyophilized, left bigger spaces
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as pores. These large pores were responsible for immediate and very high swelling ratios when 10mg+1mg of each scaffold was immersed in the PBS solution. Angiogenesis is very critical for proper and fast tissue regeneration, VEGF is very well known for stimulating angiogenesis [47], the onsite delivery of VEGF strategy could not sustain in market due to high cost and low stability. Thyroxine is one of the very promising alternative to VEGF, the sustained delivery of thyroxine to wounded area or the tissue undergoing regeneration will be helpful for fast recovery and tissue repair respectively. In current research we have observed that thyroxine was releasing from biomaterials for 7 days, on day 1 around 45% and till day 6 around 85% drug was released. This shows that these are promising materials for tissue repair and regeneration. CAM assay was used to describe the angiogenic potential of the prepared hydrogels. Minimal vessels were noticed around and inside the control scaffold, both in-vivo and ex-plant. Though blood vessels can be seen near control, little penetration had been observed in explant (Fig 9a). Maximum blood vessels were grown in and around TX-L, which was further proved by significant values for paired t-test, when compared with control both in-vivo and ex-plant (Fig 9b and c). The trend of blood vessels ingrowth decreased as the thyroxine concentration increased (TX-L > TXM > TX-H). This result helped us not only to quantify and select the best angiogenic material but also proved that thyroxine encourages the growth and penetration of endothelial cells in chitosan/carboxymethyl cellulose hydrogel at low concentrations. It is shown by ex-vivo cavity filling experiment that developed hydrogel can be administered to a cavity with the help of a syringe. It is proposed that synthesized hydrogel can be used to fill periodontal cavity either by folding around a slightly soaked piece or directly administering diluted gel in the cavity. Adhesion of diluted gel in the meat cavity in ex-vivo experiment suggested that it can remain at the site of administration for an extended period, easily. Conclusion In current research, thyroxine was added to the chitosan/carboxymethyl cellulose/hydroxyapatite hydrogels as angiogenic agent in different concentrations. The homogeneity of mixture was achieved by adding carboxymethyl cellulose powder in chitosan/HAp/thyroxine suspension. The physical characterization confirmed the presence and homogeneous distribution of all materials. 18
Hydrogels possessed interconnected porosity which resulted in rapid solution absorption and high swelling ratio. It was concluded by CAM assay that the lower concentration of thyroxine (0.1 µg) in hydrogels promoted greater angiogenesis than higher concentrations. The formation and infiltration of new blood vessels were observed in thyroxin containing ex-planted scaffolds. These studies proved that these materials are excellent candidates for periodontal regeneration.
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Acknowledgement We acknowledge Higher Education Commision project # Project# HEC-TDF-094, Pakistan Science Foundation for funding this research project# PSF-NSF/Med/P-COMSATS (01) and Ministry of Science and Technology Pakistan for financial support. We would like to acknowledge Samreen Ahtzaz and Abdur Raheem Aleem for their support and cooperation during this research work. We would also like to thank Dr Farasat Iqbal and Faisal Manzoor for their help in SEM.
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