Poly(ester amide)s from Poly(ethylene terephthalate) Waste for

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Poly (ester amide)s from Poly (ethylene terephthalate) Waste for Enhancing Bone Regeneration and Controlled Release Janeni Natarajan, Giridhar Madras, and Kaushik Chatterjee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09299 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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ACS Applied Materials & Interfaces

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Poly (ester amide)s from Poly (ethylene terephthalate) Waste for Enhancing Bone Regeneration and Controlled Release

Janeni Natarajana, Giridhar Madrasb, Kaushik Chatterjeec1

a

Centre for Nano Science and Engineering

b

c

Department of Chemical Engineering

Department of Materials Engineering

Indian Institute of Science, Bangalore-560012 INDIA

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Corresponding author Tel: 91-80-22933408. Fax: 91-80-23600472

Email address: [email protected]

ACS Paragon Plus Environment


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Abstract: The present study elucidates the facile synthesis and exceptional properties of a family of novel poly (ester amide)s (PEAs) based on bis (2- hydroxy ethylene) terephthalamide that was obtained from the poly (ethylene terephthalate) waste. FTIR and 1H-NMR were used to verify the presence of ester and amine in the polymer backbone. DSC data showed that the glass transition temperature decreased with increase in chain length of dicarboxylic acids. DMA and contact angle studies proved that the modulus values and hydrophobicity increased with increase in the chain lengths of dicarboxylic acids. In vitro hydrolytic degradation and dye release studies demonstrated that the degradation and release decreased with increase in chain lengths of dicarboxylic acids. Modeling these data illustrated that degradation and release follow first order degradation and zero order release, respectively. The in vitro cytocompatibility studies confirmed the minimal toxicity characteristic of these polymers. Osteogenic studies proved that these polymers can be highly influential in diverting the cells towards osteogenic lineage. Alizarin red staining evinced the presence of twice the amount of calcium and phosphate deposits by the cells on these polymers when compared to the control. The observed result was also corroborated by the increased expression of alkaline phosphatase. These findings were further validated by the markedly higher mRNA expressions for known osteogenic markers using RT-PCR. Therefore, these polymers efficiently promoted osteogenesis. This study demonstrates that the physical properties, degradation and release kinetics can be altered to meet the specific requirements in organ regeneration as well as facilitate simultaneous polymer resorption through control of the chain length of the monomers. The findings of this study have significant implications for designing cost effective biodegradable polymers for tissue engineering.


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Keywords: PET waste; Poly(ester amide)s; biodegradable polymers; drug delivery; tissue scaffolds

1. Introduction Despite the emergence of multitudinous biomaterials, engineering biomaterials that are capable of concomitantly eluting drugs and eliciting bone regeneration remains unfulfilled and continues to attract significant attention. Regeneration, an intricate and orchestrated biological process, will occur as a phase of repair in response to bone ailments but is severely compromised in certain clinical circumstances1. Although conventional clinical treatments in existence such as “gold standard” bone grafting exhibit relatively satisfactory results, limitations are encountered pertaining to availability and efficacy. Bone tissue engineering has been proposed to produce scaffolds as a valuable adjunct strategy to counteract these drawbacks in treating bone disorders in vivo2. Synthetic biodegradable polymers as scaffolds have emerged as an interesting discipline of research in the field of drug delivery and tissue engineering3. The influence of mechanical strength on engineering bone tissues has been extensively appreciated since the pioneering illustration of rigid substrates directing stem cells towards osteogenic lineage4. Designing polymeric biomaterials encompassing features such as biodegradability, biocompatibility, appropriate mechanical properties and possessing the potential to elute biological molecules that evoke the regeneration process constitute the pre-eminent prerequisites in recreating the physiological niche5. Owing to the depletion of fossil fuels and their fluctuating cost, there is an urgent need for the development of polymers that are sustainable and eco-friendly6. Poly (ester amide)s (PEAs) are polymers containing a combination of ester and amide blocks in the backbone of the chain7. PEAs offer an attractive combination of properties such as mechanical properties, processability and thermal properties of polyamides but degrade similar



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to polyesters7. These classes of polymers exhibit better cellular response as a result of buffering effect due to the presence of amide bonds similar to proteins8. Recently, we have reported PEAs based on soybean oil that showed excellent promise for bone tissue regeneration9. Poly(ethylene glycol) modified (PEGylated) poly(ester amide)s have been recently proposed for tissue engineering of the heart valve10. Thus, developing novel PEAs for drug delivery and tissue engineering has received significant attention in recent years. The majority of the PEAs reported have been synthesized by melt polycondensation, solution polycondensation and interfacial polycondensation but require extensive purification and possess low molecular weight compounds9, 11. An alarming societal concern is the increased accumulation of plastic waste and is leading to an environmental catastrophe12. Exhaustive usage and non-biodegradability of polyethylene terephthalate (PET) has motivated the scientists to recycle the waste13. There is also a growing demand for the manufacture of low cost biomedical polymers14. Toward this goal, recycled PET wastes have acquired wider acceptance as a feasible alternative. Chemical degradation of PET has been well established via a variety of routes such as methanolysis, glycolysis, hydrolysis, ammonolysis and aminolysis etc.13. Aminolysis has enormous potential but involves chemicals such as ethylene diamine, hydrazine and methylamine, etc., that do not yield end-products amenable for further modifications15. Aminolysis using ethanolamine is economical and requires no separation to purify the end-products15. More attractively, the end-product, bis (2- hydroxy ethylene) terephthalamide (BHETA), can be easily modified by further chemical reactions15. PET based materials have been extensively used in vascular grafts applications16. However, the non- biodegradable nature of PET makes it unfit for use as tissue engineering scaffolds. This problem can be resolved by using BHETA and has led to the development of BHETA based


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biodegradable polyurethanes17. The aromatic ring present in the structure of BHETA is expected to impart rigidity making BHETA a particularly promising material for bone tissue engineering14. Bio-inert BHETA can be converted to bio-resorbable via further processing using acids. Dicarboxylic acids (adipic, suberic, sebacic, dodecanedioic) can be used and they can be excreted by the β-oxidation pathway18. The rationale of this work was to exploit the high mechanical strength of PET positively to prepare PEAs for bone tissue engineering applications that can also be cost effective. In this framework, we present an innovative strategy to prepare PEA by reacting BHETA containing amide and - OH functional groups with several dicarboxylic acids to form ester linkages. Dicarboxylic acids with linearly increasing chain lengths were selected to obtain polymers with multifarious properties19. These linear polymers would be advantageous because they allow further modifications and enhanced solubility in numerous solvents for ease of processability. PEAs from recycled PET for biomedical applications have not been reported till date. Specifically, the physical properties, degradation and release were systematically investigated followed by the assessment of cytocompatibility. The suitability of the polymers for bone tissue engineering applications was further investigated by studying mineralization and expression of known osteogenic genes.

2. Materials and Methods 2.1 Materials Commercial PET bottles were cut into flakes of 5 × 5 mm2 followed by thorough cleansing with detergent and drying. Ethanolamine and solvent N, N dimethyl formamide were procured from Merck (India). The catalyst zinc acetate and sebacic acid were purchased from S.D. Fine



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Chemicals (India). The dicarboxylic acids such as adipic acid, suberic acid and dodecanedioic acid were obtained from Sigma Aldrich (U.S.A). 2.2 Synthesis of BHETA BHETA was synthesized based on the aminolysis of PET depolymerization with minor modifications15. 5 g of PET were taken in a 50 mL two neck round bottomed flask along with 20 mL of excess (≈1: 12 molar ratio of ET: ethanol amine) ethanol amine. 0.05 g (1 wt% of PET) (ET: Zinc acetate 98: 1 molar ratio) zinc acetate was added as catalyst to the mixture. One neck was connected to the reflux condenser. The reaction was performed in the oil bath at 180 ºC under continuous purging of nitrogen for 8 h. 100 mL of deionized water was added to the obtained product. The mixture was agitated to obtain the precipitated product (containing BHETA and dimer)17. The supernatant was decanted (containing unreacted ethanol amine)15 and deionized water was added again to the mixture and boiled to eliminate the dimer17. The supernatant was collected and cooled to ambient temperature. To ensure purity, this process was repeated for at least three times. The white precipitated powdered product, BHETA, was filtered and dried. BHETA is water soluble and will precipitate when the water mixture is cooled to room temperature. The acquired yield was around 62%. 2.3 Synthesis of PEA BHETA was further reacted with a series of dicarboxylic acids in the molar ratio of 1: 1 to obtain PEAs. BHETA and dicarboxylic acid (adipic, suberic, sebacic and dodecanedioic acids) were combined in a 50 mL round bottomed flask and the reaction was performed with uninterrupted nitrogen purging with mechanical stirring at 210 ºC for 90 min. The above reaction is a facile catalyst free melt condensation reaction with > 90% yield of a linear polymer. The reaction scheme is depicted in scheme 1.


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2.4 Nomenclature The polymers were named after the first letter of monomers. P represents polymer whereas B refers to BHETA. A is used for polymer synthesized using adipic acid, Su for suberic acid, S for sebacic acid and D for dodecanedioic acid. Thus, for example, PBSu is for polymer synthesized using BHETA and suberic acid whereas PBD was used for polymer synthesized suing BHETA and dodecanedioic acid. 2.5 Characterization of polymers 2.5.1 FTIR spectroscopy Attenuated total reflectance (mode of U-ATR) (Perkin Elmer FTIR spectrum BX) was used to acquire the FTIR spectra for the confirmation of chemical structure of the polymers. The spectra for all polymers were acquired by performing 12 scans ranging from 600 to 4000 cm-1 with a resolution of 4 cm-1. 2.5.2 NMR spectroscopy 1

H-NMR (proton nuclear magnetic resonance) was performed for PBA and PBD as

representatives using JEOL 500 MHz NMR Spectrometer. The samples (5 mg) were dissolved in d6-DMSO that was already calibrated with respect to the internal standards. The

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C-NMR

(carbon- nuclear magnetic resonance) was performed for PBD alone by dissolving 50 mg of the polymer in deuterated d6-DMSO using Bruker 400 MHZ NMR spectrometer. 2.5.3 Differential scanning calorimetry Differential scanning calorimetry (DSC, TA instruments Q 2000) was performed to acquire knowledge regarding the thermal properties of the polymers. The polymer samples (3-5 mg) were sealed to the aluminum pans. Alternate heating/cooling cycles from -50 to 200 ˚C at the



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rate of 10˚C min-1 were performed in the nitrogen atmosphere with the flow of 50 mL/ min. DSC was performed twice to obtain a consistent thermal history. 2.5.4 Determination of molecular weight The molecular weights of the polymers were calculated by gel permeation chromatography (GPC, Waters, MA), consisting of components such as pump, size-exclusion columns, a differential refractive index (RI) detector (Waters R401), and an on-line data-acquisition system. N, N Dimethyl Formamide (DMF) was used as the eluent at the flow rate of 1 mL/min. Samples dissolved in DMF were injected using a syringe in a valve (Rheodyne 7125) with a 100 µL sample loop and the refractive index was monitored and the information was stored digitally. The chromatographs were further converted to number average molecular weight (Mn) using the calibrations obtained based on polystyrene standards (Polymer Lab, Poole, UK). The molecular weight of BHETA was confirmed by performing Matrix associated laser desorption/ionization spectroscopy (MALDI TOF MS). The sample was dissolved in a mixture of Acetonitrile/ N, N Dimethyl Formamide (DMF). 2.5.5 Dynamic mechanical analysis Dynamic mechanical analysis (DMA) was employed to determine the mechanical properties of the polymers (TA, Instruments, Q 800). Polymer films of the dimension (30 mm × 5 mm × 1 mm) were cut in the room temperature (25 °C) using a cutter and were clamped to film/ fiber tension clamps and made to undergo thermal (37 °C) frequency sweep of 1 – 10 Hz at 15 µm amplitude. 0.01 N preload was conducted. Compressive tests were performed for 3D scaffolds using 15 mm compression clamps based on the above mentioned conditions. 2.5.6 Surface water wettability


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Contact angle goniometer (Data Physics) was used to characterize the surface water wettability. 1 µl ultrapure water droplet was dispensed from a syringe onto the flat polymer film. The readings were noted after the droplet attained stability. The final data are depicted as mean ± standard deviation of n = 3. 2.6 In vitro hydrolytic polymer degradation Circular discs of dimensions (4.5 mm × 1 mm) were punched using a puncher at room temperature (25 °C). These discs were placed in the nylon mesh bags and were immersed in centrifuge tubes individually containing a volume of 20 mL Phosphate buffer solution maintained at pH 7.4. These centrifuge tubes were transferred to incubator shaker operating at 37˚C and providing agitation of 100 rpm. On removal of samples at designated time intervals, water wash was performed followed by drying in a hot air oven till constant weight was achieved. The weight losses were recorded. Buffer was replaced every day to overcome the effects caused by pH differences. The % weight loss of the polymers was measured by mass differential between the initial dry weight and the weight of the samples (dry) after degrading them in buffer. The formula is, % Mass loss = (Mo- Mt) / Mo x 100

(1)

In eq. (1), Mo corresponds to the dry weight of the polymer disc before degradation commenced and Mt being the dry weight after suspending in buffer at predetermined time points. The weight losses were also recorded at various pH conditions of 3.4 and 10.4 for PBSu as a model polymer to understand the role of pH in degradation. The weight loss studies were also conducted in the presence of porcine lipase (Sigma) to understand the degradation in the presence of enzymes. 1 mg/mL lipase concentration was added to the buffer during each renewal. 2.7 In vitro hydrolytic dye and drug release



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Dyes namely, Rhodamine B, (RB, hydrophilic) and Rhodamine B base, (RBB, hydrophobic) and a hydrophilic drug, tetracycline were individually loaded in the polymers with the aim of determining drug release. The loaded concentration of the dyes and drug was 5%. The polymers were dissolved in DMF followed by addition of dye or drug to the polymer/solvent mixture. The solvent was evaporated followed by punching of the polymers. These samples were suspended in 20 mL buffer (maintained at 37°C, pH = 7.4, 100 rpm shaking) similar to degradation studies. Buffer renewal was performed every 24 h to maintain the neutral pH. 100 μL of the buffer containing the dye release products for each sample was aliquoted at predetermined intervals for a week in a well plate. The total loading concentration was calculated by submerging the samples in a large volume of NaOH to release the entrapped dye out of the polymers. A microplate reader was employed to quantify the released dye (BioTek Synergy HT, USA) by measuring the absorbance at 553 nm wavelength. The buffer containing the released tetracycline was collected and the absorbance values were read at 358 nm using a UV-Visible spectrophotometer (Shimadzu 100 Pharmaspec). Calibration curves were prepared from solutions of known concentration of the dyes and drug to determine the concentration released. The cumulative release was calculated at fixed time points and fractional release profiles were generated. 2.8 Fabrication of 3D porous scaffold 3D porous scaffolds were fabricated for PBS and PBD using the salt leaching method20. These polymers were chosen based on the higher modulus and slower degradation among the polymers studied as discussed below. 30 wt% polymer was dissolved in DMF in 2 mL centrifuge tubes. 2 g of 250- 425 µm range sieved NaCl salt was added to the tube and centrifuged at 13000 rpm for 2 min to achieve homogeneous mixing. The samples were placed in a hot air oven for 24


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h for solvent evaporation. The centrifuge tubes containing polymer/salt mixture were cut according to 8 mm diameter × 3 mm height dimensions. These samples were placed in deionized water for four days to leached out the salt with regular replacement of the water every 12 h. 2.9 Cytocompatibility of the polymer 2.9.1 Cell culture As these polymers were developed for use in bone regeneration, cytocompatibility was studied using MC3T3 E1 subclone 4 mouse calvarial pre-osteoblast cell line (ATCC, USA)21. The cells were cultured using alpha- minimum essential medium (α-MEM, Sigma, USA) augmented with 1% antibiotics (Sigma) and fetal bovine serum (10% v/v, Gibco, Life Technologies) in T-25 flasks. The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. The cells belonging to twenty third passage were detached using 0.1 mM EDTA containing 0.25% trypsin for the cytcompatibity tests. 2.9.2 Cell viability Polymer discs similar to discs used for degradation and release studies were sterilized using a combination of ethanol and UV sterilization. These discs were individually immersed in 5 mL medium in 15 mL centrifuge tubes and maintained in 37 ºC CO2 incubator for 24 h. This medium consisting of the degradation products of the polymer hereafter referred to as the conditioned medium was subsequently added to the cells to test the cytotoxicity of the polymers. Simultaneously, 2000 cells along with 200 µL culture medium were added to each well in a sterilized 96 well plate and left for 24 h before exposing them to the conditioned media. Fresh medium added to the cells served as the control. The cell viability and the cell morphology were



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indirectly analyzed following the exposure of the cells to the conditioned medium for 1 day and 3 days. Water soluble tetrazolium assay (WST-1), which measures the mitochondrial activity with the aid of oxidoreductases in viable cells, was performed to assess the cell viability after exposure to the conditioned medium22. Three wells were used for each type of polymer. 100 µL WST reagent (Roche) was added to the cells at a working concentration of 10 µL/ 100 µL medium per well. The well plate was kept in CO2 incubator for 1 h allowing the yellow color change of the media. The absorbance was measured at 440 nm using the microplate reader. 2.9.3 Live/Dead assay The cell viability was also assessed semi-quantitatively by performing the LIVE/DEAD cytotoxicity assay (Molecular probes, Invitrogen). Calcein dye in the concentration of 2 mM and ethidium homodimer in the concentration of 4 mM were used to simultaneously stain live and dead cells, respectively. The staining was performed in CO2 incubator for 15 min maintained at 37 ºC and immediately imaged in an epi-fluorescence microscope (Olympus IX-75). 2.9.4 Cell morphology For cell morphology analysis, the cells were fixed using 3.7% formaldehyde (Merck) for 15 min and imaged in bright field microscope (Olympus). The cell morphology was also examined after seeding the cells onto 3D salt leached scaffolds of PBS and PBD using a scanning electron microscope (SEM, Ultra 55, ZEISS) analysis. Gold was sputtered for 100 s to achieve a thickness of 10 nm before imaging in SEM. 2.9.5 Cell proliferation


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Cell numbers at day 14 and day 21 were evaluated by Picogreen assay (Molecular Probes) that quantifies dsDNA23. 4000 cells were seeded to the 3D scaffolds of PBS and PBD. The medium was removed and 200 µL lysis solution containing proteinase K (Sigma) at the concentration of 0.2 mg/ mL and 0.02% sodium dodecyl sulfate was added to each well in 48 well plate. This was incubated at 25 ºC for 12 h. 100 µL was taken from this solution and an equal amount of Picogreen dye (5 µg/mL in 1 X TE buffer) was added to measure the fluorescence intensity at 480 nm excitation and 520 nm emission using a microplate reader. DNA was quantified based on a calibration curve generated from serial dilutions of known DNA content. TCPS (Tissue culture polystyrene) served as controls. 2.10 Osteogenic differentiation study The cells were cultured in full culture medium supplemented with ascorbic acid (25 µM) and β glycerol phosphate (10 mM) that are known to induce osteogenic differentiation

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Osteogenic differentiation was confirmed by the calcium phosphate minerals deposited by the cells on the 3D scaffolds of PBS and PBD. 2.10.1 Alizarin red staining The calcium mineralization was quantified at day 14 and day 21 using alizarin red staining (ARS)

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(n = 3). TCPS was used as the control. The cells were fixed for 20 min using 3.7%

formaldehyde. The fixative was removed and the filtered alizarin red (1 wt%) was allowed to bind to the calcium for 25 min. The samples were washed repeatedly to remove the unbound dye. The dye bound to the calcium was dissolved using a solution containing 5% SDS in 0.5 HCl for 25 min. The absorbance values were read at 405 nm. The optical density values were normalized



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to the values of cell numbers calculated using the Picogreen assay. The presence of calcium deposits was also demonstrated by using EDX (energy dispersive X-Ray) coupled with SEM. 2.10.2 Alkaline phosphatase activity The osteogenic differentiation was also substantiated by investigating the alkaline phosphatase expression (ALP) quantitatively by using p-nitrophenyl phosphate dye (pNPP) on day 14 and day 2124. 200 µL of 0.2% Triton X- 100 was added for the cells to get lysed for 12 h. This lysis solution was freeze-thawed twice from -80 ºC to 37 ºC. 50 µL lysis solution and 50 µL pNPP dye were added and the absorbance values were acquired at 405 nm after 1 h. The values were normalized to the values of cell numbers obtained from Picogreen assay. 2.10.3 Gene expression analysis Gene expression was studied by quantitative analysis of mRNA of Run related transcription factor 2 (Runx2), bone morphogenic protein (BMP-2), osteocalcin (Bglap) and osteopontin (Spp1) genes using quantitative real time-PCR (RT-PCR). β- Actin (GAPDH) was used as the house- keeping gene. The primer sequences are tabulated in Table 1. The isolation of RNA from MC3T3 E1 cells cultured on 3D scaffolds of PBS and PBD on day 14 was performed using RNA isolation miniprep kit (Agilent) following the protocol provided by the supplier. The total RNA extracted was checked for quality and quantity by obtaining O.D. at 260 and 280 using a microplate reader. 10 µL RNA was totally reverse transcribed to obtain cDNA using Highcapacity cDNA reverse transcription kit (Applied Biosystems). RT-PCR was performed using 5 ng cDNA in 10 µL PCR reaction (1X Sybr, Kappa) and primers (Integrated DNA Technologies). The PCR reactions were performed in three steps: 95 ºC for 50 s, 95 ºC for 15 s for 40 cycles and 60 ºC for 15 s. Data were processed by 2-∆∆Ct method to calculate the fold changes with respect to


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the internal control (house- keeping gene). The experimental data are presented as average ± standard deviation of three independent experiments with triplicates. Statistical Analysis: The error bars in figures represent standard deviation. The significant differences were evaluated by using one way ANOVA along with Tukey’s test. Significance levels were set to be p < 0.05.

3. Results and discussion 3.1 Polymer synthesis BHETA is formed by the reflux condensation of ethanol amine with zinc acetate as the catalyst. As nitrogen is more electronegative than oxygen, the ester link of PET will be attacked by the amine group present in the ethanol amine15. Sodium acetate was shown to be a better catalyst in this seminal study15. Many studies claim zinc acetate to be a better catalyst among all metal catalysts for PET recycle reactions where maximum yields up to 65% are reported25. Therefore, in the present study, the reaction has been slightly modified by utilizing zinc acetate as the catalyst. Zinc acetate will accelerate the reaction by possibly forming complex with carbonyl group and thus, increasing the polarity15, 26. The molar ratio of ET: ethanolamine was taken in excess to ensure complete reaction. 1: 6 molar ratio of ET: ethanolamine is reported in previous studies17 but the molar ratio taken in this study was 1: 12. Similarly, 1 wt% zinc acetate (98: 1 molar ratio of zinc acetate: ET)25 was used because 1 wt% seems to provide maximum yield irrespective of the type of metal catalysts15. Furthermore, 8 h reaction was proven to be efficient because increasing the time may lead to the degradation of amide15, 25. 180 ˚C to 196 ˚C was proven to be the effective temperatures for zinc acetate25. Therefore, the reaction was conducted at 180 ˚C. The molecular weight of BHETA was confirmed before proceeding to the polymerization step. The reaction between BHETA and dicarboxylic acid is conventional



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esterification with water as the byproduct resulting in the formation of ester. No usage of cytotoxic catalysts was involved that could be detrimental to the use of the polymers as biomedical materials. The ratio of monomers was maintained at 1: 1 ratio for a complete reaction of chemically active moieties. The polymers were soluble in common solvents such as DMSO and DMF. The reaction scheme for poly (BHETA dodecanedioate) is portrayed in Scheme 1. 3.2 Polymer characterization 3.2.1 FTIR analysis The chemical structure of the polymers was confirmed by FTIR. FTIR spectra27 (Figure 1) of BHETA and polymers confirmed the presence of amide, –OH and ester groups. The peaks at 3280 cm-1 and 1530 cm-1 correspond to secondary amine stretch and bend, respectively. The peak at 1630 cm-1 could be attributed to carbonyl (C=O) group stretching of amide bond. The peak at 1260 cm-1 could be ascribed to aromatic C-N stretch. The peaks at 3364 cm-1 and 1050 cm-1 in the FTIR spectra of BHETA could be associated with primary alcohol (-OH) stretch and C-O stretch of (CH2-OH), respectively. The characteristic ester peak around 1721 cm-1 associated with (C=O) carbonyl stretching was observed in all polymers indicating that alcohols of BHETA have completely reacted with acid groups of dicarboxylic acids. It could be noted that the peaks related to –OH were also absent in the FTIR spectra of the polymers. Other peaks such as C-C ring stretch at 1500 cm-1 and symmetric and symmetric C-H stretching at 2940 cm-1 and 2840 cm-1 can be observed in all spectra. The spectra confirmed the formation of BHETA and PEAs. 3.2.2 1H and 13C- NMR spectroscopy The chemical compositions of BHETA and the PEAs were further verified by performing 1

H-NMR and

13

C- NMR spectroscopy (Figures 2a-2d). In 1H-NMR spectra of BHETA (Figure

2a), the peaks present around 3.5 ppm correspond to the aliphatic methylene protons near –OH


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group15. The peak at 3.1 ppm can be correlated to the methylene protons adjacent to –NH group. The peak at 4.7 ppm can be correlated to the protons of –OH group. The peak at 7.9 ppm can be attributed to the aromatic protons. The peaks at 8.5 ppm may be ascribed to the amide group protons. The peaks and their integral values match with the previously reported literature15, 28. Figures 2b and 2c represent the 1H-NMR spectroscopy of PBA and PBD29. The peaks between 3.5 to 4.0 ppm correspond to the aliphatic methylene protons similar to Figure 2a. Similarly, the peaks around 7.9 ppm and 8.5 ppm represent the aromatic protons and protons of amide group, respectively. It should be noticed that the peak at 4.7 ppm of BHETA is absent in these spectra indicating that –OH groups have reacted to form esters. The peak at 2.5 ppm may be correlated to the residual DMSO. The peaks between 1.0-2.2 ppm belong to the dicarboxylic acids. The peak at 2.2 ppm can be ascribed to the methylene protons next to the ester group (O=C-CH2). The peak at 1.5 ppm is attributed to the methylene protons adjacent to those next to ester groups (O=C-CH2-CH2). The peak at 1.2 ppm can be assigned to the inner most methylene protons. This peak did not show up in PBA spectra since adipic acid does not have these methylene groups. In addition to the 1H-NMR,

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C- NMR spectra of PBD is shown (Figure 2d). The peaks

between 25-35 ppm can be matched to the aliphatic methylene carbons of the polymer whereas the peaks at 128 ppm can be correlated to the aromatic carbons. The peak at 165 ppm is ascribed to the carbon atoms of the amide group whereas the peak at 175 ppm can be assigned to the carbon atoms of the ester groups. 3.2.3 Differential scanning calorimetry



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The thermal characterization by DSC (Table 2, Figure S1) revealed the amorphous nature of the polymers. No melting (Tm) or crystallization peaks (Tc) were observed. The glass transition temperature (Tg) was determined based on the integration of the DSC curves. There was an appreciable decrease in Tg with an increase in chain length of the dicarboxylic acids. The highest Tg was observed for PBA that was 53º whereas the PBD exhibited the lowest Tg of 36º among the polymers studied. There is an increment in aliphatic content with increase in chain lengths of the dicarboxylic acids. This would contribute to the increased amorphous nature resulting in higher free volume. Higher free volume results in lower Tg30. When 1,4 butane diol was reacted with succinic and adipic acid, the ester based on adipic acid had lower Tg31. Similar decrease in Tg was observed for PEAs when a series of increasing chain lengths of diamine was reacted with glycerol and sebacic acid32. PBA and PBSu were glassy at the physiological temperature (37 oC). The overall trend for Tg can be written as: PBA> PBSu> PBS> PBD 3.2.4 Determination of molecular weight The molecular weight of BHETA was confirmed by MALDI TOF MS which was 252 Daltons. The peak at 268 Daltons corresponds to the reaction intermediate before the rearrangement reaction when PET is reacted with ethanolamine, as given in the schematic for the aminolysis of PET 15 (Figure S2). GPC showed the exponential increase in molecular weight of these PEAs with increase in chain length of the dicarboxylic acids9 (Figures S3a-S3d). The number average molecular weights of PBA and PBSu are 18000 and 38000, respectively (Table 2). However, as the end of the polymer peak is near the start of the solvent peak in the GPC chromatograph, these molecular weights may been slightly erroneous (± 2000). The Mn of PBS and PBD are 1600000 and 290000, respectively (Table 2). The polydispersity of all the polymers was approximately 1.2 ± 0.2. When phenyl alanine and allyl glycine were reacted with various


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diacids and diols with increase in methylene groups to form PEAs, Mn increased from 21000 to 39000 g/mol33. Several studies have reported the increase in molecular weight with increase in chain length (increase in methylene groups) of diols or diacids7. Increase in molecular weight based on increase in methylene groups depends on numerous factors such as pKa values, steric effect and inductive coupling. The pKa values increase with increase in chain lengths of dicarboxylic acids. They are 4.41, 4.53, 4.72 and 4.9 for adipic acid, suberic acid, sebacic acid and dodecanedioic acid, respectively. Lower the pKa, stronger is the acid and the adjacent electron withdrawing substituents increase the acidity by further stabilizing the carboxylate. Resonance stabilization of the carboxylate ion allows the negative charge to be delocalized between the two electronegative oxygen atoms. Therefore, increasing chain lengths leads to a decrease in acidity and the resonance stabilization leads to higher reactivity. This leads to an increase in the molecular weight of the polymer formed. Steric hindrance increases with molecular size that induces electronic repulsion between non-bonded atoms of reacting molecules leading to higher reactivity. The inductive effect results from the increase in electronreleasing ability of the acid with lengthening alkyl chain and facilitates the protonation of the carbonyl oxygen34. This protonation leads to an increase in reactivity that result in the higher molecular weight formation for longer chain lengths of the dicarboxylic acids. When glucitol was reacted with diacids with increasing chain lengths, the polymers composed of monomers with shorter chain lengths degraded faster because of lower molecular weights34. It can be noted that the molecular weights of these polymers were exceptionally high compared to many reported PEAs with molecular weights ranging between 10000-600008, 35. The molecular weight of PBD was 7.6 times higher than that of PBA. Aromatic groups present in the polymers will contribute to the higher molecular weight17. Thus, with the change in chain lengths of the



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monomers, it was possible to gain tremendous variations in molecular weight. The trend for molecular weights can be presented as follows: PBD> PBS> PBSu> PBA 3.2.5 Dynamic mechanical analysis The Young’s modulus values were calculated from the following formula E * = E ' 2 + E "2

(2)

where E*, E’2, E’’2 corresponds to the complex modulus, storage modulus and loss modulus, respectively. The complex modulus is considered as Young’s modulus in many cases36. Mechanical characterization by DMA (Table 2) indicated the increase in the values of Young’s modulus with increase in chain lengths of the dicarboxylic acids as the molecular weights of the polymers increase37. The modulus values ranged from ranging between 30 to 130 MPa. The modulus value was the lowest for PBA and the highest for PBD among the polymers studies here. The increase in modulus values was approximately 4.5 times from PBA to PBD. A similar increase of 3.0 to 11.5 MPa was observed when dissimilar amines with varying chain lengths were reacted with sebacic acid and glycerol32. In the reaction of maltitol with suberic and dodecanedioic acid, the modulus increased from 12 to 368 MPa for the same molar ratios, respectively22. These values were comparable to the previously reported crosslinked PEAs when soybean oil and diethylene triamine were reacted with different chain lengths of dicarboxylic acids where the polymer based on dodecanedioic acid possessed the modulus of 120 MPa9. Increase in hydrophobicity results in higher modulus9. The presence of bulky aromatic groups also elevates modulus to a greater extent14. Similar increase in the compressive modulus was observed with the increase in molecular weight in the case of castor oil based polyanhydrides18. These linear PEAs from PET in contrast to the crosslinked PEAs from soybean oil can not only


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be synthesized faster but afford markedly enhanced processability for the preparation of 3D scaffolds and encapsulation of drugs. As evident from Table 2, there were wide variations in modulus values with the variations of chain lengths of the monomers. These modulus values are comparable to the modulus values of both hard and soft tissues. For example, the modulus value of elastin, knee cartilage and aorta are 1.1 MPa, 11.8 MPa and 6.0 MPa14, 38. Owing to a good match with modulus of soft tissues, PBA can be applied for soft tissue regeneration. The modulus of cancellous bone ranges from 50-100 MPa39 suggesting that the other polymers of this study could be used for repair and regeneration of bone tissue. These polymers have modulus higher than several other biodegradable polymers such as poly(lactic-co-glycolic acid) and polycaprolactone approved for clinical use whose modulus are reported to be just 3 MPa and 1 MPa, respectively38. The trend of the modulus values can be represented as PBD> PBS> PBSu> PBA. Because of the inability to test the moduli of the samples in tensile mode due to their soft nature, compressive tests were performed and it revealed that the modulus of the 3D scaffolds of PBS and PBD were 22 kPa and 28 kPa, respectively. The values were higher than that of the 3D salt leached scaffolds of polycaprolactone40. 3.2.6 Contact angle measurements The biological outcome of the material and the polymer degradation are known to be largely influenced by the hydrophobicity of the surface14. Contact angle measurements demonstrated the hydrophobic nature of these polymers (Table 2). Contact angles showed a significant increase with the increase in chain lengths of the dicarboxylic acids. The values are in the range of 82º to 100º. As the hydrophobicity increases with methylene groups, the contact angles also increase. Similar increase was observed when galactitol was reacted with a series of dicarboxylic acids



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with the same molar ratios19. When erythritol was reacted with sebacic acid and dodecanedioic acid, contact angles showed an increase from 58° to 80°41. The trend for contact angles is: PBD> PBS> PBSu> PBA. The contact angles for the 3D scaffolds were also measured since the surface porosity influences the wettability of the material. The contact angles were found out to be 82° and 89° for PBS and PBD, respectively. This was lesser than that of the flat polymer films of PBS and PBD as porosity can increase the hydrophilicity of the material and thereby, enhance the cellular response42. 3.3 In vitro polymer degradation 3.3.1 In vitro hydrolytic polymer degradation and kinetics Understanding the degradation behavior of the polymer is crucial for tissue engineering applications because the time scale of the degradation of the scaffold and the regeneration of the tissue should match for best clinical outcomes. The degradation of the polymers was studied by (a) measuring the mass loss and (b) measuring the loss in molecular weight by GPC. With respect to the hydrolytic degradation (Figure 3a), the fastest degrading polymer was PBA with 21% mass loss over a week. This was followed by PBSu and PBS exhibiting 9% and 4% weight losses in a week, respectively. The slowest degrading polymer was PBD with a negligible mass loss of 2% in a week. When the degradation study was extended till 21 d, PBA, PBSu and PBS demonstrated 46%, 24% and 9% loss, respectively. The lowest degradation was exhibited by PBD with just 6% weight loss in 21 days. The trend can be written as: PBA> PBSu> PBS> PBD. The loss in molecular weight was recorded after 21 days. GPC illustrated the decrease in molecular weight to 9600 from 18000 for PBA. Similarly, PBSu and PBS showed an appreciable


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decrease in molecular weights to 29500 and 131000, respectively, from the original molecular weights listed in Table 2. The molecular weight of PBD reduced to 220000 from 290000. The molecular weight of PBA decreased almost to half of the initial molecular weight since it is the fastest degrading polymer in this study. Hydrophobicity and modulus play critical roles in dictating the degradation behavior. With an increase in hydrophobicity and modulus, the degradation of the polymers decreases. Hydrophobic polymers display slower degradation in an aqueous medium (PBS). Generally, aromatic polymers exhibit slower degradation because of the bulky group43. Contact angle demonstrated the decrease in hydrophilicity with increase in chain lengths of the dicarboxylic acids. The modulus value and molecular weight increased with increase in chain lengths of diacids. Therefore, the degradation of the polymers reduced with increase in modulus, contact angle and molecular weight. In previous reports, the degradation of the polymers showed a significant reduction with increase in chain lengths. When glycerol and threitol were reacted with poly (1,3 diamino 2- hydroxy propane), the polymers exhibited 97% and 70% weight losses with glycerol and threitol in a week, respectively44. Similarly, when ethane diol and butane diol were reacted with 1,4 butane diol and biasmide diol, the mass losses were 5.7% and 3.6% in 5 days, respectively45. In soybean oil based PEAs, the degradation decreased from 100% to 7% in 24 h with increase in chain lengths of the dicarboxylic acids9. The molecular weights decreased from 70000 to 60000 in 28 days when l- phenyl alanine was reacted with butane diol and sebacoyl chloride8. The decrease in molecular weights of these polymers was comparable to the previous reports8. The power law was employed to model the kinetics of the degradation21,



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−

=

(3)

In eq. (3), M denotes the polymer mass, t represents the time, kd corresponds to the degradation rate constant and n signifies the order. The plots were fitted with n=1 signifying first order degradation. The insets of Figure 3a show the linear plot obtained when – ln (Mt/M∞) is plotted with time. The slopes resulted in kd values and they are tabulated in Table 3 with intercept being zero. Based on rate constant values, it is apparent that PBA degraded fastest among all polymers. PBA degraded more than two times faster than PBSu, six times faster than PBS and twelve times faster than PBD. The degradation rate constant value decreased from 13.5 × 10-4 to 1.2 × 10-4 from PBA to PBD. The trend of the rate constant of degradation was similar to the trend of the degradation. Thus, the chain length can be used to tune the degradation kinetics of the polymers to tailor their use for various biomedical applications. 3.3.2 In vitro hydrolytic degradation of polymers in different pH It is important to understand the effect of micro-environment on the degradation of the polymers as these polymers are proposed to be used as implants for a wide range of biomedical applications. pH can vary widely in different organs of the human body. Digestive system exhibits acidic pH46 whereas wound sites in chronic inflammation possess basic pH47. Amides are cleaved only in the presence of enzymes48. Therefore, ester hydrolysis is the contributing factor for the hydrolytic degradation of PEAs. Basic pH accelerates the degradation of the esters49. PBSu served as a model for this study given its moderate degradation in this family of polymers. The degradation was recorded in acidic (3.4) and basic mileus (9.4) (Figure 3b). No appreciable differences were observed in the degradation of the polymer in acidic and neutral


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pH. However, faster degradation occurred in basic pH, as expected. The polymer exhibited 48% weight loss in a week in basic pH whereas only 9% and 8% weight losses were recorded in neutral and acidic pH, respectively (Figure 3b). Degradation at different pH was also modeled using Eq. 4 and it followed first order kinetics (Inset of Figure 3b) with the kd values being 31.9 × 10-4 h-1 and 4.6 × 10-4 h-1 for basic and acidic pH, respectively. The rate of degradation in basic medium was almost seven times faster than acidic and neutral medium. 3.3.3 In vitro hydrolytic degradation of polymers with porcine lipase Lipases are the enzymes that are involved in the hydrolysis of esters in our body. Conventionally, the degradation of the polymers is faster in enzymatic medium50. In the case of these PEAs, the magnitude of the differences in degradation was slightly higher in enzymatic medium than the hydrolytic environment, as anticipated. PBA degraded 25% in a week, whereas 15%, 11% and 6% weight losses were observed for PBSu, PBS and PBD in a week, respectively (Figure 3c). The polymers based on L-alanine, sebacic acid and butane diol exhibited 77% weight loss in the presence of enzyme whereas only 66% weight loss was observed in the absence of enzyme50. Similarly, methionine based PEAs degraded faster in the presence of chymotrypsin8. The degradation of the polymers in the enzymatic medium also followed first order kinetics with PBA degrading six times faster than PBD (Inset of figure 3c). When compared to the degradation in the absence of lipase, many of these polymers degraded almost three times faster in enzymatic medium (Table 3). 3.4 In vitro release kinetics 3.4.1 In vitro dye release kinetics



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Biomolecules such as growth factors and drugs are often incorporated in the polymeric scaffolds to augment the bioactivity through controlled release of the molecules23. Drug delivery systems decrease systemic drug toxicity, improve target specificity and protect the drugs against biochemical degradation7. The release kinetics are governed by myriad features such as hydrophobicity, diffusion, degradation of the material and dispersion of drugs inside the material, etc.18,

51

The trends of the release for both the dyes were similar to that of the

degradation (Figure 4a and 4b). For RB (Figure 4a), PBA showed the highest release of 18% in one week. This was followed by PBSu and PBS with 9% and 4% release, respectively. PBD showed the lowest release of just 2% in a week. With respect to RBB (Figure 4b), the trend was similar to that of RB release. PBA and PBSu showed a similar release of 18% and 9% in a week in comparison to RB, respectively. However, PBS and PBD showed lesser release of 3% and 1.5% in a week in comparison to RB, respectively. Similar trends of dye release was observed with increasing chain lengths of dicarboxylic acids in soybean oil based PEAs. Approximately 20% release of the dyes was observed from dodecanedioic acid based PEAs which is similar to PBA9. The trend of RB release and RBB release is as follows: PBA> PBSu> PBS> PBD. In previous cases, many polymers demonstrate two phases in release. In the first phase, there will be a rapid outburst of dyes followed by a second phase of controlled release. The portion of the dye that does not blend with the polymer contributes to the burst release52. When the polymer is surrounded by PBS, the unblended dye on the surface gets released abruptly in the initial phase. Later, the sustained release will occur in which the encapsulated dye is released. There will be formation of holes and channels once the polymer starts degrading. This will encourage the entry of the water inside the polymer bulk. In this family of polymers, no burst release was observed.


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The degradation of the polymer played a vital role in regulating the release since the trends of the degradation and release were similar. Hydrophobicity, moduli and molecular weight determined the kinetics of the degradation. The water inflow triggered the release of the dyes implying the strong role of diffusion. The slower release of hydrophobic RBB in comparison to hydrophilic RB can be attributed to the diffusion11. The dye release was modeled based on Korsmeyer-Peppas model, a semi empirical relation53 used to study the drug dissolution from polymeric systems. The equation is written as

= (4)

In eq (4), Mt and M∞ signify the amount of the dye released at the specified time period t and at infinite time, respectively. The slope k represents the rate constant whereas n denotes the release exponent intimating of the possible mechanism of the dye transport across the polymer. This equation is valid only for initial 60% of the release profile53. In order to determine the release exponent and the rate constant, Mt/M∞ vs time was plotted on a log-log plot and n was found to be 1. When n = 0.5 it represents Higuchi kinetics/Fickian diffusion and n = 1 is zero order release. The linear plot of ln Mt/M∞ versus ln t when n = 1 are shown as insets of Figure 4a and 4b. The slopes (k) were calculated from the initial points of the lines with intercept zero (Table 3). It should be noted that the data had a best fit for n =1 with R2 values greater than 0.9. As evident from the rate constants, in the case of RB and RBB, the rates of PBA were two times, six times and twelve times approximately higher than PBSu, PBS and PBD, respectively. This variation elucidates that the release rates can be modulated based on changing the chain lengths of monomers befitting rapid or sustained release applications. For example, rapid release is favored in treatment of wounds54 whereas slower controlled release is favored in treating



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inflammations55. PBA can be used for quick release applications whereas PBD can be employed for sustained release applications. Controlled release is preferred to prevent the concentration of the drugs rising to toxic level. These polymers displayed only controlled release suggesting their potential use in various release applications. 3.4.2 In vitro drug release kinetics Tetracycline, a hydrophilic antibiotic, was loaded inside the polymers and the release kinetics was studied. The trend and the kinetics of the tetracycline release were similar to the dye release. PBA showed the highest release of 20% whereas the PBD showed the lowest release of 2%. The rate constant for PBA was 2.5 times, 5 times and 12 times higher than that of PBSu, PBS and PBD, respectively. Drugs like ibuprofen and TEMPO were loaded in PEAs and approximately 40% release of the drugs was achieved within a month52, 56. This is comparable to the release of tetracycline achieved from these polymers. 3.5 Fabrication of 3D porous scaffold Owing to their slow degradation and higher modulus, salt leached scaffolds of PBS and PBD were prepared for bone tissue engineering. SEM images (Figure 5) revealed the porous nature of the scaffolds and the pores were observed to be interconnected when compared to the SEM images obtained from flat films of PBD (Figures. S4a and S4b). The average pore size was ~150 µm. Pre-osteoblasts were subsequently seeded on the porous scaffolds to study the osteogenic differentiation, as described below. 3.6 Cytocompatibility studies 3.6.1 Quantitative assessment of cell viability by WST assay


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Studying the cytocompatibility characteristics of these polymers that are developed for biomedical applications is of primary importance. Given that these polymers exhibit modulus comparable to that of the bone components, MC3T3-E1 mouse pre-osteoblasts were exploited to assess their capability in aiding the cell growth. WST assay was used to evaluate the viability of the cells. The live cells are solely responsible for converting tetrazolium salts to water soluble yellow formazan crystals. The absorbance values are the representation of the quantity of viable cells that were gauged based on a calibration curve. All PEAs were tested for cytocompatibility. The assay proclaimed the minimally cytotoxic nature of these polymers (Figure 6). Day 1 data of the polymers showed excellent cell attachment on all PEAs. The number of viable cells was higher for control (TCPS) when compared to PEAs with the differences being statistically significant. The cell numbers were similar for all PEAs on day 1 with no statistical differences. The increase in cell numbers was more than twice for all PEAs from day 1 to day 3 suggesting the cyto- friendly nature of these PEAs. PBS and PBD showed similar cell numbers to that of TCPS on day 3 and the differences were not statistically significant. However, the differences in cell numbers for PBA and PBSu with respect to TCPS were statistically significant. This could be attributed to their fast degrading nature when compared to PBS and PBD. Accumulation of degradation products due to rapid degradation tends to increase the acidity of the conditioned medium that can affect cell viability. However, the polymers are unlikely to be toxic in vivo where the continuous flow of bodily fluids will minimize local accumulation of the degradation products. 3.6.2 Qualitative assessment of cell viability by Live/Dead assay The qualitative assessment of cell viability was achieved by performing concurrent Live/Dead assay. The images substantiated the data of WST assay (Figure 7). Control (TCPS)



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demonstrated the highest fraction of live (green) cells. The fraction of viable cells looked similar for all PEAs with PBA exhibiting modest reduction. The percentage of viable cells was approximately 95% in all polymers. These micrographs revealed that the cells were metabolically active and further confirmed that the polymers exhibit minimal cytotoxicity. 3.6.3 Cell morphology analysis Cell morphology was analyzed since it is indicative of cell function57. Bright field optical micrographs (Figures 8a and 8b) revealed that the cells displayed the characteristic “spindle shaped” morphology. No differences in morphology could be observed between the cells treated with the fresh medium and the conditioned medium of PBD. The cells appeared to be healthy well spread and exhibited good cell-cell communication with filopodial protrusions. It should be noted that the morphology and the viability studies were conducted after the exposure of the cells to conditioned media. One can conclude that the degradation products appear to be non-toxic to the cells and did not seem to alter cell morphology markedly. 3.6.4 Cell proliferation assessment by DNA quantification The quantification of DNA content can be correlated to the cell numbers. The total DNA content increased from day 14 to day 21 on the 3D porous scaffolds of PBS and PBD (Figure 9). The DNA content was higher for TCPS than the scaffolds likely because of the hydrophobic nature of the polymers. The differences between the samples and TCPS were statistically significant on both day 14 and day 21. However, the increase in DNA content in both the polymers proves that the polymers support cell attachment to the scaffolds and proliferation thereafter. No discernable differences in DNA contents were observed between PBS and PBD at


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day 14 or day 21. Although the DNA content on the PEA scaffolds was lower than the TCPS control, they exhibited good ostegenic ability as described below. 3.7 Osteogenic differentiation study 3.7.1 Mineralization by alizarin red staining In vitro osteogenesis studies were conducted to test the ability of the polymers to augment bone formation in vivo for potential clinical use. The deposition of calcium minerals by cells on the surface of PBS and PBD scaffolds was quantified by AR staining (Figure 10). AR staining revealed that these scaffolds exhibited markedly higher absorbance values compared to TCPS on both day 14 and day 21. The differences in calcium deposition between the control and the samples were statistically significant. Increase in calcium deposition was observed in the scaffolds and control from day 14 and day 21. Although there were no statistical differences in absorbance values between PBS and PBD on day 14, the differences were significant on day 21. SEM micrographs revealed rounded cellular morphology that can be attributed to the surface hydrophobicity58 (Figure 11a). Greater calcium deposition on the surface of PBD was further evidenced by EDX (16 wt%) (Figures 11b and 11c). Therefore, it can be inferred that these polymers are remarkably efficient in inducing osteogenesis. Protein adsorption plays a key role in mediating the interactions between the cells and a biomaterial59. Properties such as charge on the polymer surface, chemical functional groups and surface wettability profoundly determine the formation of the adsorbed protein layer59. Amide groups are known to promote osteoblast attachment and proliferation60. Amine and carboxyl groups on the surface of the material participate in the orientation of the proteins’ conformation to facilitate integrin binding in osteoblasts thereby activating the osteogenic differentiation



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signaling pathways61. Previous reports also suggest that polymer surfaces rich in amine and carboxyl groups are effective in binding calcium and phosphate ions to promote osteogenesis62. Surfaces modified with amine groups exhibit enhanced osteogenic activity in the presence as well as absence of soluble osteogenic factors63. The 3D PEA scaffolds are rich in amine groups, which could have contributed to the observed in vitro osteogenic activity. The higher modulus and slower degradation of PBD compared to PBS could have contributed to its superior osteogenic ability. 3.7.2 Alkaline phosphatase expression For further independent validation of osteogenesis, the expression of ALP was assayed. ALP expression by the cells is considered as an early marker of osteogenic differentiation64. Figure 12 shows that ALP expression on either scaffold was higher than TCPS confirming enhanced osteogenic activity of the polymeric scaffolds. Note that the ALP activity decreased from day 14 to day 21 on the scaffolds. However, the activity increased in TCPS from day 14 to day 21. The differences in absorbance values were statistically significant for PBS and PBD in comparison to TCPS on both day 14 and day 21.

Similar to ARS, there were statistical

differences in absorbance values between PBS and PBD on day 21. This could also be ascribed to the higher modulus of PBD. Since ALP is an early marker, the ALP expression likely decreased on the scaffolds from day 14 to day 21 after attaining the maximal expression. Similar decrease in ALP expression has been reported previously65. ALP expression increases initially and may decrease with time to be followed by mineralization. However, in the case of TCPS, where the expression was low initially, the value continued to rise as days progressed.


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ALP data corroborate the results of mineral deposition confirming that the PEA scaffolds possess excellent osteogenic ability and are thus promising candidates for bone tissue engineering. PBD showed improved osteogenic characteristics when compared to PBS. Higher modulus and slower degradation should be credited for the improved osteogenic quality of PBD. Nevertheless, both polymers demonstrated remarkable osteogenic capability compared to TCPS. These polymers can be further tested in vivo for eventual clinical use for bone tissue engineering. 3.7.3 Gene expression analysis The changes in the expression of mRNA of some known osteogenic markers were studied to further confirm osteogenesis. Because cells on PBD and PBS showed higher mineralization when compared to those on the control, gene expression studies were performed to corroborate the mineralization result. Osteoblast differentiation is comprised of three sequential processes namely proliferation, maturation of the extracellular matrix and mineralization. The genes related to these processes are expressed at every time point and are highly specific to osteoblasts66. Various activation and suppression signals result in driving progenitor cells to osteogenic lineage24. Runx2 plays a critical role in bone development and repair. Widely known as a core binding factor (cbfa1), it is an early marker for osteoblast differentiation24. The expression of Runx2 stimulates the bone specific genes associated with the differentiation of osteoprogenitor cells to bone lineage and also initiates mineralization24,

67

. BMP-2 (bone morphogenic protein), a

member of TGF-β family, is an ECM matrix secreted by osteoblasts. It is a marker related to osteoblast differentiation and skeletal development24, 67. BMP-2 activates Runx2 and Alpl and subsequently, the differentiation process is activated by both Runx2 and BMP-267. Osteocalcin



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(OC), otherwise known as bone gamma carboxy glutamic acid protein (bglap)24, is a late marker expressed during mineralization24. Osteopontin (OP) , also known as Spp124, is again a late marker related to bone development and resorption67. On day 14, the relative expression of mRNA was significantly higher for PBS in the case of OC and OP when compared to control (Figure 13). The expression for Runx2 and BMP-2 in the case of PBS did not differ significantly compared to control. The expression of mRNA was significantly higher for PBD in comparison to the control for all the genes studied. PBD showed higher levels of mRNA expression for all genes when compared to PBS. There was a two fold increase for Runx2, BMP-2 and OP expressions in the case of PBD when compared to control. Similarly, the expression of OC in the case of PBD was three fold times higher compared to control. Materials containing –OH and –NH2 moieties on their surfaces revealed up-regulation of genes associated with osteogenesis61. Thus, the data of mRNA expression served as an additional proof to validate the mineralization studies.

4. Summary and conclusions The current study provides valuable insights regarding the effectiveness of this simple novel family of PEAs based on recycled PET and dicarboxylic acids for potential biomedical applications. Importantly, this study also elucidates that physical properties, degradation, release and cellular responses were hugely influenced by the variations in the chain lengths of the dicarboxylic acids. The observed changes in the physical properties can be summarized as follows: a)

Glass transition temperature showed a significant reduction with increase in chain length

of the dicarboxylic acids.


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b)

Modulus values and contact angles increased with increase in chain lengths of the

dicarboxylic acids. c)

The degradation and release also revealed a decrease with increase in chain lengths of the

dicarboxylic acids. The degradation and release followed first order kinetics and zero order release, respectively. Therefore, this study highlights that the aforementioned properties can be modulated by careful selection of the monomers to tailor the properties for specific biomedical applications. Moreover, the polymers can also be converted into thermosets by the addition of more monomers and crosslinking them could result in polymers with enhanced modulus. Viability assays confirmed the cytocompatibility of these polymers. PBA possessed a modulus of 30 MPa but exhibited a faster degradation of 20% in a week. This can be used in applications requiring rapid release. PBS and PBD exhibited optimal moduli of 120 and 130 MPa matching the modulus of cancellous bone and slower degradation of 4% and 2% weight losses in a week. The osteogenic differentiation and gene expression studies demonstrate that these polymers are excellent candidates for bone tissue regeneration that provide suitable mechanical cues and biomimetic niche.

Acknowledgements This

work

was

funded

by the Department

of Biotechnology (DBT),

India

(BT/PR5977/MED/32/242/2012). G.M acknowledges J.C. Bose fellowship from DST, India. We would like to thank Ms. Queeny Dasgupta of Center for Biosystems and Engineering for DSC and DMA analysis. J.N. would like to thank Mr. Sumit Bahl, Dr. L.R. Jaidev and Dr. Jafar Hasan of Dept. of Materials Engineering for technical assistance regarding contact angle analysis and



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36

cell studies. Mr. Sai Rama Krishna Meka is gratefully acknowledged for help with PCR work. We would also like to thank NMR Research Centre of IISc regarding NMR analysis. We would also like to acknowledge Molecular Bio Physics Unit (MBU, IISc) for MALDI TOF MS analysis.

Associated content: Supporting information (SI): DSC thermograms of all PEAs (Figure S1), MALDI TOF MS spectra of BHETA (Figure S2) GPC chromatogram showing the variation of RI detector response with retention time for PBA (Figure S3 (a)), GPC chromatogram showing the variation of RI detector response with retention time for PBSu (Figure S3 (b)), GPC chromatogram showing the variation of RI detector response with retention time for PBS (Figure S3 (c)), GPC chromatogram showing the variation of RI detector response with retention time for PBD (Figure S3 (d)), SEM image of surface of PBD at 9 KX. Scale bar represents 1 µm (Figure S4 (a)), SEM image of surface of PBD at 200 X. Scale bar represents 100 µm (Figure S4 (b))


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42

Table 1. Primer sequences for RT-PCR Gene β-actin

Primer sequences (F-forward, R-reverse) F:5′-TCTTGGGTATGGAATCCTGTG-3′

Size (bp)

Ref

82

68

90

68

151

68

149

68

239

69

R:5′-AGGTCTTTACGGATGTCAACG-3′ Runx2

F:5′-AAGTGCGGTGCAAACTTTCT-3′ R:5′-TCTCGGTGGCTGGTAGTGA-3′

Bmp-2

F:5′-GGTCACAGATAAGGCCATTGC-3′ R:5′-GAGGACAGGGAGGATCAAGT-3′

Bglap

F:5′-TGCTTGTGACGAGCTATCAG-3′ R:5′-GAGGACAGGGAGGATCAAGT-3′

Spp1

F:5′-ACA CTT TCA CTC CAA TCG TCC-3′ R:5′-TGC CCT TTC CGT TGT TGT CC-3′


Page 43 of 64

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43

Table 2. Physical properties of the synthesized PEAs Polymers

Tg (ºC)

Mn (Daltons)

Young’s

Contact

modulus

angle (º)

at 10 Hz (MPa)

PBA

53

18 x 103

29.6

82 ± 1

PBSu

45

38 x 103

94.6

85 ± 2

PBS

38

16 x 104

120.7

90 ± 3

PBD

36

29 x 104

130.2

100 ± 2



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Table 3. Degradation and release rate coefficients of PEAs Hydrolytic

Enzymatic

RB

RBB

Tetracycline

degradation,

degradation,

Release,

Release,

Release,

rate

rate

kRB, h-n

kRBB, h-n

kTET, h-n

coefficient

coefficient

(× 10-4)

(× 10-4)

(× 10-4)

(h-1) (kd)

(h-1) (kd)

( × 10-4)

( × 10-4)

PBA

13.5

18.7

12.8

11.9

12.1

PBSu

5.6

10.1

6.8

6.7

5.2

PBS

2.1

7.1

2.5

1.8

2.3

PBD

1.2

3.5

1.5

1.1

1.2

Polymers


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45

Figure captions Scheme 1. Reaction scheme for Poly (BHETA-Dodecanedioate) Figure 1. FTIR spectra of BHETA and all PEAs Figure 2. NMR spectra of (a) BHETA (1H ) (b) PBA (1H ) (c) PBD (1H ) (d) PBD (13C) Figure 3. In vitro degradation profiles of the PEAs in 20 mL PBS solution (a) Hydrolytic degradation of all PEAs at pH = 7.4 (b) Hydrolytic degradation of PBSu at different pH (c) Hydrolytic degradation of all PEAs at pH = 7.4 in the presence of porcine lipase. The insets show the variation of – ln (Mt/M0) with time and the degradation rate constants are determined from the slopes of the linear plots and are tabulated in Table 3. Figure 4. In vitro release profiles from all PEAs in 20 mL PBS solution (pH = 7.4). Release of (a) RB (b) RBB (c) Tetracycline. The insets of all the plots show the variation of Mt/M∞ with t and the release rate constants, kRB, kRBB and kTET are obtained from the slopes of the linear plot and are tabulated in Table 3. Figure 5. SEM image of 3D salt leached scaffold of PBD at 200 x magnification. Scale bar indicates 200 µm. Figure 6. Cell viability of PEAs determined by WST assay for day 1 and day 3. * above the bars indicate that the samples are statistically significant when compared to control. Figure 7. Fluorescent images of Live/Dead viability assay performed for PEAs on day 3 (a) PBA (b) PBSu. (c) PBS (d) PBD (e) TCPS. Scale bar indicates 20 µm. All images are taken at 4 x magnification. Viable cells appear green while non- viable cells appear red. Figure 8. Optical micrographs of MC3T3 E1 cells (a) treated with fresh medium and (b) treated with medium containing the degradation products of PBD at 10 x magnification. Scale bar is 20 µm Figure 9. DNA quantification of MC3T3 E1 cells cultured on 3D scaffolds of PBS and PBD by Picogreen assay on day 14 and day 21. * indicates that the samples are statistically significant (p < 0.05) when compared to control. The results represent mean ± SD for n = 3. Figure 10. Quantification of mineral deposition by ARS dye on PBS and PBD scaffolds on Day 14 and Day 21. * indicates that the samples are statistically significant (p < 0.05) when compared to control. # represents the statistical differences across the samples. The results represent mean ± SD for n = 3. Figure 11. SEM micrograph coupled with EDS spectra of mineral deposited polymer scaffolds on Day 14. Arrow marks show the round morphology of the cells (a) SEM micrograph of PBD



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(b) EDS spectra of PBD (Magnification = 400 X, scale bar = 100 µm). (c) Atomic weight percentage of different elements obtained from EDS spectra. Figure 12. Quantification of ALP expression of PBS and PBD scaffolds on Day 14 and Day 21. * indicates that the samples are statistically significant (p < 0.05) when compared to control. # represents the statistical differences across the samples. The results represent mean ± SD for n = 3. Figure 13. Relative mRNA expression obtained by RT-PCR on PBS and PBD scaffolds on day 14 normalized to control (TCPS). * indicates that the samples are statistically significant (p < 0.05) when compared to control. # represents the statistical differences across the samples. The results represent mean ± SD for n = 3.


Page 47 of 64

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47

Scheme 1

HO

CH2

CH2

NH

O

O

C

C

NH

CH2

CH2 OH

BHETA

+

O

OH HO O Dodecanedioic acid

210oC, 2 h N2 atm

O

O

C

C

O

O

* O

CH2

CH2

NH

NH

CH2

CH2

O O

*

n

O Poly (BHETA-Dodecanedioate)



48

4000

BHETA

1721 1630 1530 1500 1260 1050 860

2940 2840

3364 3280

Figure 1

% Transmittance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 64

PBA

PBSu PBS

PBD

3500

3000

2500

2000

1500

Wavenumber (cm-1)


1000

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49

Figure 2

HO

CH2

CH2

NH

3

2

1

5

O

O

C

C

NH

CH2

CH2

OH

1

2

3

5 4 2

4

(a)

BHETA

1

5 3

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0


2.5

2.0

1.5

0.28 0.36

7.0

0.20 0.26 0.04

7.5

0.13

8.0

775.29 6.79 3.49

8.5

1.29

9.0

2.10

9.5

4.12

10.0

2.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

0.5

ppm


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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O

O

4 5 O2

CH2 CH2 NH C

1

3

O

O

O

5 4

C

NH CH2 CH2 O

3

2

5 4

4 5 O

1

(b)

5

4 3

10

1

8

6

4

ppm


2

0

Page 51 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


51

O

5 6 6

6 4

*

4 6 6 6 5

O

O

O

O

CH2 CH2 NH C

C

1

1

3

NH CH2 CH2 O

3

2

5 6 6 6 4

O

1

1

*

6 6 6 5

4

O

2

(c)

6

1

5 4

3

10

8

6

4

ppm


2

0

n


52

O

CH2 CH2 NH C

C

NH CH2 CH2 O

3

1

137.37

173.85 173.48

1

166.72

4

3

1

O

2

*

O

1

4

40.70 40.49 40.28 40.07 39.86 38.42 36.25 34.57 34.36 31.55 29.71 29.60 29.34 26.17 25.40 25.28

*

63.29 63.03

O

130.16 128.33 128.04

O

4

O

O

4

207.59

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 64

PBD

(d)

1

2

4 3

210

200

190

180

170

160

150

140

130

120

110

100

90

80

70


60

50

40

30

20

10

0

ppm

Page 53 of 64

53

Figure 3

50

% Weight loss

0.6

- ln (M t /M 0 )

40

0.2

0.0

30

(a)

0.4

0

100 200 300 400 500 Time (h)

PBA PBSu PBS PBD

20 10 0 0

100

200

300

400

500

Time (h)

0.6

% Weight loss

40

(b)

0.4

-ln (Mt/M0)

30

0.2

0.0

0

24 48 72 96 120 144 168 Time (h)

pH 3.4 pH 7.4 pH 10.4

20

10

0

0

24

48

72

96

120

144

168

Time (h)

30

(c)

0.3

- ln (M t/M 0 )

% Weight loss

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

0.2

0.1

0.0

0

24 48 72 96 120 144 168 Time (h)

PBA PBSu PBS PBD

10

0 0

24

48

72

96

120

144

Time (h)


168


54

Figure 4

(a)

0.20 0.15 M t/M ∞

Fractional release

0.20

0.10 0.05

0.15

0.00

0

24 48 72 96 120 144 168 Time (h)

PBA PBSu PBS PBD

0.10

0.05

0.00 0

24

48

72

96

120

144

168

Time (h)

(b)

0.2

Mt/M∝

Fractional release

0.20 0.1

0.15 0.0

0

24 48

PBA PBSu PBS PBD

0.10

72 96 120 144 168 Time (h)

0.05

0.00 0

24

48

72

96

120

144

168

Time (h)

0.25

(c)

0.20

Mt/M ∝

0.2

Fractional release

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 64

0.15

0.1

0.0

0

24 48 72 96 120 144 168 Time (h)

PBA PBSu PBS PBD

0.10 0.05 0.00 0

24

48

72

96

120

144

Time (h)


168

Page 55 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55

Figure 5



56

Figure 6

Day 1 Day 3

10000

Number of cells

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 64

8000 6000

*

*

4000 *

*

*

PBSu

PBS

*

2000 0 PBA

PBD

Polymers


TCPS

Page 57 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


57

Figure 7

(a)

(b)

(c)

(d)

(e)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 58 of 64

58

Figure 8

(a)

(b)


Page 59 of 64

59

Figure 9

3500 Day 14 Day 21

3000 2500

DNA (ng)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2000 1500

*

*

*

*

1000 500 0 PBS

PBD

Polymers


TCPS


60

Figure 10

Mineral staining (Abs405/DNA (ng))

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 60 of 64

0.0005

# *

Day 14 Day 21

0.0004 *

0.0003 *

0.0002 *

0.0001 0.0000 PBS

PBD

Polymers


TCPS

Page 61 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


61

Figure 11

Element

Weight%

Atomic%

CK

48.23

60.48

OK

35.49

33.41

Ca K

16.28

6.12

Totals

100.00



62

Figure 12

0.012

ALP activity (Abs405/ DNA (ng))

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 62 of 64

0.010

Day 14 Day 21

*

*

# *

0.008 0.006

*

0.004 0.002 0.000 PBS

PBD

Polymers


TCPS

Page 63 of 64

63

Fold changes of mRNA expression with respect to control

Figure 13

#

PBS PBD

*

3

# 2

*

# *

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


# *

*

1

0 RunX-2

BMP-2

OC

Genes


OP


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 64 of 64

64

Graphical abstract

Poly (ester amide)s developed from recycled PET exhibited tailored drug release, excellent cytocompatibility and osteogenesis.


Poly(ester amide)s from Poly(ethylene terephthalate) Waste for

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