Novel Fabricating Process for Porous Polyglycolic Acid Scaffolds by

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Novel fabricating process for porous polyglycolic acid scaffolds by melt-foaming using supercritical carbon dioxide Jiapeng Zhang, Shengbing Yang, Xi Yang, Zhenhao Xi, Ling Zhao, Lian Cen, Eryi Lu, and Ying Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00692 • Publication Date (Web): 26 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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ACS Biomaterials Science & Engineering

Novel fabricating process for porous polyglycolic acid scaffolds by melt-foaming using supercritical carbon dioxide Jiapeng Zhang,1 Shengbing Yang,2 Xi Yang,3 Zhenhao Xi,*, 1 Ling Zhao,1 Lian Cen,*,1 Eryi Lu,4 Ying Yang5

1. Shanghai Key Laboratory of Multiphase Materials Chemical Engineering, East China University of Science and Technology, No. 130, Meilong Road, Shanghai, China 2. Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedics Surgery, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, No. 639, Zhizaoju Road, Shanghai, China 3. Department of Plastic and Reconstructive Surgery, Shanghai 9th People's Hospital, Shanghai Jiaotong University School of Medicine, No. 639, Zhizaoju Road, Shanghai, China 4. Department of Stomatology of Renji Hospital, School of Medicine, Shanghai Jiaotong University, No.160, Pujian Road, Shanghai, China 5. Institution of Science and Technology in Medicine, University of Keele, Hartshill Stoke-on-Trent, ST4 7QB UK Email: [email protected] Email: [email protected] 1

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Email: [email protected] *Email: [email protected] Email: [email protected] *Email: [email protected] Email: [email protected] Email: [email protected]

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Abstract Polyglycolic acid (PGA) is a biocompatible and biodegradable polymer with high crystallinity. It is difficult to obtain PGA porous scaffolds with controllable morphology as well as outstanding mechanical properties without toxic solvents. The current study thus aimed to develop a novel melt-foaming strategy to prepare porous PGA scaffolds through the interaction between PGA molecules and supercritical carbon dioxide (scCO2). Before the design of foaming strategy, rheological properties of PGA were first studied by a Haake rheometer, while the effect of scCO2 on PGA was investigated by high-pressure differential scanning calorimetry (DSC). It was revealed that the elasticity and viscosity could be greatly improved by a temperature regulation operation to withstand the growth of bubbles at the initial depressurization. Meanwhile, the melting and crystallization temperatures of PGA were reduced due to the plasticization effect of scCO2. Through the dissolution of compressed CO2 into PGA melt and subsequently rapid depressurization at a relative low temperature with high PGA melt strength, PGA scaffolds with porosity of 39–74%, average pore sizes ranging from 5 to 50µm, and interconnectivity over 90% could be controllably fabricated. The effect of foaming temperature and pressure on morphology of PGA foams were then explored in detail. Special nano–scale morphology on the pore surface of resultant porous PGA foams was observed. These PGA foams also exhibited attractive compressive modulus of 68-116MPa. The PGA foams with 74% porosity and average pore size of 38µm, prepared at 208oC and 20MPa were then used as scaffolds for in vitro cellular evaluation. Fibroblasts seeded on the 3



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scaffold exhibited excellent spreading shape and good proliferation ability and in vivo implantation of PGA foams manifested as the notable tissue ingrowth and neovascularization process within the foams, ascertaining its potential applications for tissue engineering and regenerative medicine. This work presents a breakthrough to fabricate highly crystalline PGA into porous scaffolds instead of traditional fibrous ones.

KEYWORDS: Polyglycolic acid, High crystallinity, Supercritical carbon dioxide foaming, New strategy, Porous scaffold

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1. Introduction Polyglycolic acid (PGA) is a semi–crystalline aliphatic polyester with good mechanical and biocompatible properties.1-2 It is non–toxic and degrades in the body via hydrolysis, yielding glycolic acid (GA) or GA oligomers as degradation products,3 while GA can be further decomposed in the carbohydrate cycle within the body.4 Hence, it has been approved by FDA for implantation in the human body.5 In the field of medical technology, PGA as well as its co– polymers has been successfully applied as wound closure materials, surgical sutures, controlled drug delivery carriers, and bone fixation devices in forms of pins, rods, plates and screws.4, 6-11 Not only in immunocompromised experiments,12-14 recent research have further substantiated its potential as scaffold materials for tissue engineering with promising results even in immuno–competent animal studies.15,

16

For example, PGA fibers and autologous

adipose–derived stem cells (ASCs) were used to repair Achilles tendon defects in rabbits.15 The same fibers were also used for urethral reconstruction in a canine urethral defect model when seeded with ASCs combined with oral mucosal epithelial cells.16 Fibrous PGA scaffolds have even been commercialized (Biofelt®, Cellon S.A., Luxembourg), demonstrating their high biocompatibility and potential clinical application.

Despite its wide applications both in commercialized medical products and ongoing research, the processing of PGA has mainly relied on few traditional molding technologies including extrusion, injection and spinning.17 Conventional manufacturing approaches to 5



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fabricate tissue engineering porous scaffolds, such as solvent casting–salt leaching, do not apply to PGA, due to the fact that PGA with a high crystallinity (45–55%) is insoluble in most organic solvents18-19 except the highly toxic hexafluoroisopropanol (HFIP). Consequently, it is difficult to obtain PGA porous scaffolds with tunable porosity and pore size as well as distribution of pores. Most of the current developed PGA scaffolds are based on PGA nonwoven fibers produced by melt–spinning, which have poor compressive properties. Therefore, development of a new fabricating process for porous PGA scaffolds with controllable morphology and mechanical properties is of importance for medical and regenerative medicine applications.

Supercritical technique is a completely organic solvent free process in which supercritical fluids, such as supercritical carbon dioxide (scCO2) which is nontoxic, inexpensive, nonflammable and is able to easily reach its supercritical point with large solubility in polymers,20-21 is applied as an efficient, green and physical foaming agent. The rapid depressurization or temperature rise at a relatively low temperature leads to the formation of foam structure comprised of processing polymers. This technique has been used to produce porous foaming materials,22 drug or protein release carriers,23-24 and nano–particles.25-26 The success of foaming strategy depends highly on the choice of processing parameters, such as saturation temperature, duration and pressure, based on investigations on interactions between scCO2 and polymers, plasticization and crystallization behaviors of polymers.27 Consequently, 6


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different structures could also be yielded to meet various engineering requirements. For example, bi–modal bubble structure of polystyrene (PS) was obtained by a two–step batch foaming process,28 while sandwich–structure of polyethylene glycol terephthalate (PET) microcellular foams could be fabricated by controlling scCO2 diffusion and induced crystallization behavior.29 However, the solid-state scCO2 process cannot be simply applied to highly crystalline PGA which has high melting point and easily degrades. Mooney et al attempted to foam PGA as scaffolds for tissue engineering in order to avoid the usage of toxic organic solvents, and it turned out that no evidence of pore formation in PGA materials could be observed when applying the routine scCO2 process (solid-state).30 By contrast, this protocol is easy to produce porous polylactic acid (PLA) and polycaprolactone (PCL) scaffolds.30-31-32

To overcome the high crystallinity barrier and obtain porous PGA scaffold, a new scCO2 assisted foaming process was developed in this work on the basis of detailed investigations on the effects of CO2 on the melting and crystallization behavior of PGA. The working principle of this novel foaming technique was that the supercritical fluid, scCO2, was dissolved into PGA melt in the saturation state as a foaming agent to fabricate controllable porous scaffold after rapid depressurization at a relatively low temperature with high PGA melt mechanical strength. The effective range of foaming temperature and CO2 pressure have been investigated in detail. The morphology and apparent properties of the resulting foamed PGA matrix were then characterized, followed by the subsequent cellular performance of the PGA foams as scaffolds 7



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for human fibroblasts. The histocompatibility of these PGA scaffolds was characterized by subcutaneous implantation in a rat model. It is believed that the current study introduces a novel and promising supercritical foaming process for preparing PGA porous foams with tunable porosity and sufficient mechanical strength. This is an important breakthrough in PGA processing for tissue engineering or regenerative medicine application. The temperature and pressure range explored in this work offers a valuable reference to further develop PGA medical devices.

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2. Experimental 2.1 Materials PGA granules were purchased from Corbion Purac (PURASORB PG S, 1.4dL/g, Netherlands). The melting temperature measured by differential scanning calorimeter (DSC) was 232oC in nitrogen at ambient pressure.

Dispase and collagenase were from Worthington (Lakewood, NJ). Fetal bovine serum (FBS) was acquired from Hyclone (USA). Dulbecco’s Modified Eagle Medium (DMEM) was from Invitrogen (USA). L–glutamine, vitamin C, penicillin, DAPI and streptomycin were all from Sigma–Aldrich (USA). Phalloidine solution was from Cytoskeleton (USA). CCK8 kit was from Dojindo (Japan). 2.2 Rheological measurements Rheological properties of PGA samples were measured using a Haake MARS III Rheometer (Thermo Fisher Scientific, USA) in a parallel disk mode under nitrogen. The PGA granules were first dried in vacuum at 30oC for 3h and at 110oC for 9h successively, and then compression molded at 190oC into 35mm (diameter) ×2mm (thickness) disks. Subsequently, the disks were heated to 237oC for 5min to make sure that crystals were melted. The angular frequencies, ω, were swept from 102 to 10–1rad/s at different temperatures of 230, 220, 215 and 210oC. Complex shear viscosity η*, storage modulus G’ and loss modulus G’’ as a function of angle frequency were determined under a constant stress of 10Pa. 9



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2.3 Thermal analysis A high–pressure DSC (NETZSCH 204 HP, Germany) was used to study thermal properties of PGA granules and PGA foams, especially the melting temperature and crystallization behaviors, both in N2 and compressed CO2 atmosphere. The calibration of DSC calorimeter was carried out by determining the fusion of In, Bi, Sn, Pb, and Zn under ambient and high pressures of CO2, respectively.33 An aluminum crucible loaded with a mass of 12mg unprocessed PGA specimens or PGA foams were placed in the DSC chamber. After being swept by CO2 three times, the chamber was pressured by CO2 up to a desired value (0.5–6MPa) at 190oC for 1h. Thereafter, the chamber was heated to 235oC at a heating rate of 10oC/min and held for 10 min to eliminate any prior thermal history before being cooled to 160oC at a cooling rate of 1oC/min to investigate PGA crystallization behavior. Afterwards, the cooled PGA was again heated to 235oC at 10oC/min for plasticization effect study. The melting temperature, Tm, and the melt crystallization temperature, Tc, (the enthalpy of crystallization,

) at different

compressed pressure were determined from the DSC curve, respectively.

To determine the crystallinity of raw PGA specimens and obtained PGA foams, samples were measured by DSC under N2 atmosphere as described above. The crystallinity was calculated by the following equation,34

(1)

10


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where

was the enthalpy of crystallization per gram and

was the enthalpy of

crystallization per gram of 100% PGA crystallinity. For unprocessed PGA particles,

was

76.31 J/g.34 2.4 Apparatus and foaming process A high–pressure vessel equipment with an electronically temperature–controller, a needle valve (SS-1RS8MM-A, Swagelok, USA) and a ball valve (SS–1RS8MM, Swagelok, USA) were tailor–made to perform the batch foaming process. In foaming procedures, the opening angle of needle valve was set constant and kept same for all samples. Whilst during the depressurization, the ball valve was opened fully to achieve the rapid and maximum depressurization. The temperature was measured with a calibrated thermocouple. The vessel pressure was measured at an accuracy of ±0.01MPa by the pressure transducer (P31, Beijing Endress & Hauser Ripenss Instrumentation Co., Ltd.) The CO2 loading was achieved by a syringe pump (DZB–1A, Beijing Satellite Instrument Co., China), with an accuracy of 0.01cm3.

After loading the high–pressure vessel with 150 mg PGA granules in an iron mold (4 cylinders; inner diameter: 10mm; height: 10mm), the foaming process was initiated by sweeping the chamber with CO2 for three times, and the vessel under CO2 atmosphere of 0.3MPa was heated by a hot oil bath to the temperature (Tmelt) to melt PGA. It took approximately 10min for the temperature in the chamber to reach the desired value. During this 11



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period, a predetermined amount of CO2 was charged into the vessel to reach a certain pressure, called as the pressure at melting temperature (Pmelt) when the temperature was maintained at Tmelt. The vessel was maintained at this state for 15min for equilibrium. The temperature was subsequently declined to the saturation temperature (Tsat), while Pmelt was raised to the saturation pressure (Psat) simultaneously. The vessel was maintained at this state for another 15min to ensure that the whole system was in a stable state. Afterwards, the valve was opened to release the compressed CO2 rapidly so as to induce nucleation and bubble growth in the melt PGA. The Tsat and Psat were varied to investigate their respective effects on foaming. The samples were retrieved from the high-pressure chamber immediately after depressurization, and cooled by the CO2 expansion and spontaneously by room temperature. 2.5 Morphology observation analysis The porous morphology of PGA foams was characterized using scanning electron microscopy (SEM, NOVA Nano 450, FEI, USA). The foam specimens were immersed in liquid nitrogen (–196oC) for 10min and then fractured to create the cross–section for examination. The fractured surfaces were imaged after platinum (Pt) coating. The pore size distribution was obtained through the SEM image analysis by the Image–Pro Plus software (Media Cybernetics, Silver Spring, MD). The average number for the diameter of pores (D) was calculated using the following equation based on the SEM micrograph:

(2) 12


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The apparent densities of PGA foams ( ) were measured referring to the ASTM standard (D792–00)

35

by weighing polymer foams with a sinker and calculated using the following

equation:

(3)

where ρwater is the density of water as 1 g/cm3, m is the apparent mass of sample in air without the sinker, m’ is the apparent mass of sample and the sinker completely immersed in water, and M is the apparent mass of the entirely immersed sinker. The expansion ratio of PGA scaffolds, Rv, is the ratio of the bulk density of unprocessed PGA particles ( ) to that of the foamed one ( ):

(4)

The porosity of foamed PGA (p), a fraction of the volume of voids over the total volume, was determined by the following equation:

(5) 2.6 Interconnectivity analysis The interconnectivity of PGA foams was investigated by a true volume and density measurement instrument (1200e, Quantachrome, USA). The tests were carried out under nitrogen atmosphere of 117kPa at room temperature. The operational mode was a flowing

13



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procedure for 10min per run and 10–time replicates were taken to obtain the mean value with a deviation of 0.03cm3 correspondingly. The interconnectivity of PGA foams (I) was calculated by the following equation:

×

where Vopen

bubble

(6)

is the volume of open bubbles directly given by the instrument and

! ""

# $ is the previous outcome according to ASTM standard (D792–00). %

2.7 Crystal form analysis The crystal forms were measured by X–ray diffractometer (D8 ADVANCE XRD, Bruker–AXS, Germany) with a Cu–Kα radiation at 40kV and 40mA. After vacuum drying at 110oC for 12hours, the PGA particles and foams were then smashed to the size of less than 75µm. The XRD spectra were collected using 2 theta (θ) range of 10o to 90o at a scanning speed of 10o/min in a continuous scan mode. The crystal size (D) was calculated by the Scherrer formula: '(

& ) *+, -

(7)

in which K was the Scherrer constant, B was the full width of half maximum in XRD patterns, θ was the diffraction angle, and γ was the wave length of X ray, 0.15nm.

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2.8 Mechanical properties Mechanical properties of PGA foams (5×5×3mm) were measured by an INSTRON tester (INSTRON 5542, USA) with a crosshead speed of 0.6mm/min and load cell of 500N at room temperature.36 The compressive load-displacement plots were generated by the software, Instron Bluehill. The displacements of 0.05 mm were chosen from the initial point at linear part of the curves as elastic regions for compressive modulus calculation of all samples using the formula:

.

/ ⁄0 ∆3⁄3

(8)

where F is force, S is the sample area, h is sample height. The average values of modulus with standard deviations were presented from five samples for each experimental condition. 2.9 Cell source Human skin fibroblasts were used in this study, which was approved by the ethics committee of Shanghai Ninth People's Hospital Affiliated School of Medicine of Shanghai Jiao Tong University and informed consent from all of the patients were obtained. Fresh human foreskin specimens were obtained from donors (aged from 5 to 12 years) who received a routine circumcision procedure at Shanghai Children’s Hospital, China. The specimens were washed with sterile phosphate–buffered saline (PBS), and cut into small pieces (1–2mm3) which were then digested with 0.1% dispase at 4°C overnight. The epidermal layers were removed, and the remaining dermal parts were further digested with 0.1% collagenase in 15



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DMEM for 3h by gentle agitation at 37°C. The digested cells were then forced to pass through a 100µm cell strainer (BD Biosciences, USA) and further centrifuged (Allegra 64R Centrifuge, Beckman Coulter, California, USA) at 1500rpm for 5min. The cells were collected by resuspension in low–glucose DMEM supplemented with 10% FBS, L–glutamine (300mg/mL), vitamin C (50mg/mL), penicillin (100U/mL), and streptomycin (100mg/mL). After the cell suspension was plated for 24h, the plates were washed thoroughly with PBS to remove residual non–adherent cells and replaced with fresh media for further culture. When 90% confluence was reached, cells were detached and subcultured at 1×104 cells/cm2 in the culture plates. For the following experiments, cells of passage 3–5 were used. 2.10 Cell culture onto PGA scaffolds The non–porous skin of PGA foam was removed to prepare homogenous PGA scaffolds (7×7×1mm) for cell culture. The PGA scaffolds were sterilized by gamma ray at 20kGy via Shanghai Heming Ltd. Cells were harvested with trypsin–EDTA treatment, centrifuged and re–suspended in the growth medium. Aliquots of cell suspensions, 20µL, were then evenly seeded into the PGA scaffolds with a seeding density of 5×104 cells/scaffold. The cell–scaffold constructs were then placed in wells of culture plates for 4h to allow cell attachment. After that, each construct was transferred to a fresh well and 1ml of the growth medium was then added into each well of 24 well plates. The medium was changed every other day. After being incubated for predetermined time durations, the cell–scaffold constructs were harvested for the 16


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following analyses. 2.11 Cell–scaffold construct morphology Fibroblast adhesion and extracellular matrix (ECM) deposition onto the scaffolds was investigated by a scanning electron microscope (NOVA NanoSEM450, FEI, USA). Briefly, the cell–scaffold constructs were carefully removed from the culture medium, and then washed twice with PBS followed by being fixed with 0.25% glutaraldehyde for 10h at 4oC. After that, the samples were washed with PBS for three times, dehydrated through an ethanol series, and then dried using a critical point drier (HITACHI HCP–2, Japan). The dried samples were mounted, sputter–coated with Pt, and then viewed under SEM.

The growth and distribution of fibroblasts on PGA scaffolds were further visualized by a confocal laser scanning microscope (CLSM Germany, Leica TCS SP8). After careful washing, the phalloidine solution (5µg/mL) was added to the cell–scaffold constructs for 30min. The scaffolds were further washed with PBS solution for 3 more times, and incubated with DAPI for 15min. After being washed with PBS thoroughly, the constructs were observed by CLSM. 2.12 Cell proliferation assay Cell proliferation was determined by CCK–8 assay according to the manufacture’s protocol. Briefly, the constructs after being incubated for 1, 3, and 7 days were added with 50µL of CCK8 solution for 2–hour incubation at 37°C. The absorbance of the culture media in each well was measured at 450nm using a plate reader (Thermo Scientific Varioskan Flash, 17



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Finland). Cells seeded on the wells directly without scaffolds were used as the control group. The viable cell number of each sample was measured by CCK-8 kit and calculated through the standard curve. 2.13 In vivo subcutaneous implantation of PGA scaffolds in rats The experimental protocol involving animals was approved by the Animal Care and Experiment Committee of Shanghai Jiao Tong University School of Medicine. Scaffolds (7×7×3 mm) were sterilized by gamma–ray of 20kGy and then surgically implanted into the hypodermis of the back of 9 adult male Sprague–Dawley rats (weighing around 100g). The surgical procedures started with anaesthesia by injecting 1% pentobarbital sodium of 400µL into cavum abdominis. The dorsal area of a rat was then shaved and a longitudinal skin incision (1cm) was made subcutaneously on the back using an ophthalmic scissor. The subcutaneous tissue was separated from the full skin layer to form a cavity. Each cavity was inserted with one PGA foam. After the successful insertion, the incision was sutured. Tissue specimens were obtained from the three implants performed in each of the three animals, sacrificed on days 7, 14, 21 and 28 days. 2.14 Histological analysis of subcutaneous implanted PGA scaffolds After fixation with 4% neutral buffered formalin, the extracted tissue–scaffolds were dehydrated in a graded series of alcohol, and then embedded in paraffin. Serial 4µm thick sections were stained with hematoxylin and eosin (H&E), and observed microscopically. 18


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3. Results and Discussion 3.1 Design of foaming strategy for PGA The key issues in PGA foaming are its high crystallinity at ambient temperature and low viscoelasticity at its melting temperature. Since foaming mainly induces melt stretching, it is of importance to evaluate such melt rheological properties for PGA. Hence, the dynamic rheological measurements were first performed to simulate and instruct such a theoretical temperature–decline practice.

5

4

10

10

4

10

210 C o 215 C o 220 C o 230 C

3

10

3

10 G'/ Pa

η */ Pas

o

o

210 C 2

10

o

1

10

0

-1

10

0

10 -1 ω/ rad⋅s

1

10

215 C o 220 C o 230 C

10 -2 10

2

10 -2 10

2

10

-1

10

0

1

10

10

2

10

-1

ω/ rad⋅s

5

10

4

10 G''/ Pa

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


3

10

o

210 C o 215 C o 220 C o 230 C

2

10

-2

10

10

-1

0

10

10

1

2

10

-1

ω/ rad⋅s

Figure 1. η* (a), G’ (b) and G’’ (c) versus frequency (ω) for PGA at different temperatures. Under ambient N2, the chamber temperature was heated to 237oC to melt PGA, and then cooled to the test temperature. 19



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The complex viscosities (η*), storage modulus (G’), loss modulus (G’’), and loss factor (tanδ) of PGA melt at different testing temperature as a function of frequency (ω) are shown in Figure 1. As illustrated in Figure 1a, the complex viscosities were found to gradually increase with the decline in the test temperature from 230oC to 210oC at the same frequency. A slight shearing thinning behavior of PGA melt appeared at 230oC. Similar tendency of such shearing thinning behavior could also be observed on PGA at either 220oC or 215oC. However, when the temperature was decreased to 210oC, the complex viscosity of PGA displayed a relatively more obvious shearing thinning phenomenon, and the η* values kept on decreasing with the increase in ω to the one that was close to the value of 215oC at the frequency of 102rad/s.

Analogously, the storage modulus, G’, of PGA at 230oC, 220oC, and 215oC all increased dramatically with the increase in frequency from 10–1 to 102rad/s, as shown in Figure 1b. As for the G’ of PGA at 210oC, it seemed that the values reached a plateau at the low frequency range (10–1–100rad/s) without obvious dependence on frequencies. Upon the further increase in frequencies from 100rad/s, similar tendency of G’ was observed at 210oC to those of other test temperatures. At the same frequency, G’ was found to gradually increase with the decline in the test temperature from 230oC to 210oC. The obvious rise in G’ from 215oC to 210oC within the low frequency region (10–1–100 rad/s) suggested that the elastic property of molten PGA was greatly reinforced at 210oC.

The loss modulus of PGA, G’’, all increased with the increase in frequency at different 20


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testing temperatures, as shown in Figure 1c. Similar dependence of G’’ on frequency and temperatures could be observed to that of G’, except that the plateau phenomenon at the low frequency range at 210oC was not so evident. The fact that G’’ was also enhanced in the lower temperature suggested that the PGA melt could relatively better withstand the shear strain.

PGA can be melted at its melting point and then quenched to a lower temperature for foaming at a stable molten state. The obtained rheological behaviors over temperature, stronger viscoelasticity of PGA melt at lower temperature, were thus of importance to have a significant effect on melt foaming using scCO2 as higher elasticity, G’ and viscosity, G’’, could provide better resistance to bubble collapse in the foaming process. Moreover, it was shown that elasticity could be better strengthened than viscosity within the low frequency region and the shearing thinning phenomenon of PGA melt was more apparent. Considering

4 ∗ 678 9 :; + 78 99 :; />, in general, this rheological behavior over temperature was more pronounced in G’ than in G’’ within 10–1–100rad/s, in consistence with the above observed phenomena.

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Figure 2. DSC thermograms of PGA and diagram of PGA foaming procedure. (a) Melting temperature (Tm) at ambient N2 or compressed CO2; (b) Crystallization temperature (Tc) at ambient N2 or compressed CO2; (c) Foaming procedure developed in this work.

Remarkably, in the presence of CO2, the interaction between CO2 and polymer should affect the thermal properties of PGA as long as compressed CO2 dissolved into the polymer. Subsequently, high pressure DSC was deployed to study such course. The DSC curves of Tm and Tc were plotted against different CO2 pressure at a cooling rate of 1oC/min as shown in Figure 2a and b. The melting temperature was approximately 232oC under N2 atmosphere. The melting temperature gradually decreased with the increase in the compressed CO2 pressure. 22


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The temperature difference reached –5oC when the compressed CO2 was pressurized from normal atmosphere pressure to 6MPa. On the other hand, the cooling DSC curve also shifted to the low–temperature side with the increase in CO2 pressure, indicating that the dissolution of CO2 in PGA matrix effectively postponed the crystallization of PGA melts. Under ambient N2, the crystallization temperature was 200oC which could be declined to 196.5oC under CO2 atmosphere of 6MPa. The linear relationship in the investigated conditions between ∆Tm and CO2 pressure, as well as that between ∆Tc and CO2 pressure, generalized from the DSC patterns, could be established in the following:

, (R=0.99)

, (R=0.99)

(8)

(9)

with a standard deviation of 0.005oC and 0.05oC, respectively.

The melting temperature could be altered by compressed CO2, as compressed CO2 could act as a plasticizer in the saturation process.37-39 In other words, the dissolved CO2 induced PGA to swell with an increased free volume between the molecular chains. After the saturation process, the mobility of molecular chains of PGA was improved when the melting temperature was depressed. During this process, the crystallization temperature decreased with increasing pressure due to the aforementioned mechanism. The temperature range of Tc–Tc (P) could be indicated by the PGA crystalline behavior at lower temperature induced by compressed CO2. 23



Page 24 of 52

Hence, essentially, with a decreasing melting temperature, PGA crystals could be melted at a lower temperature which was favorable in constituting the system equilibrium and avoiding thermal degradation at the same time. Meanwhile, in the presence of scCO2, melt–state foaming interval could be extended to a lower temperature in which PGA was amorphous with better rheological properties rather than semi–crystalline. In brief, the interaction between CO2 and PGA could realize the energy conservation and extend the operation region. However, the current DSC instrument could not be performed at pressures higher than 6MPa, hence we could not simulate the whole foaming process through the DSC measurements.

With full view on the rheological behaviors over temperature and thermal properties over compressed CO2 on PGA, a novel foaming strategy was thus designed based on the fact that PGA could be completely melted to erase the crystals for saturation and then kept in the molten state in a lower temperature range for foaming.

Taking plasticization effect and the heat transfer force into consideration also, the operation conditions of the saturation pressure and temperature could generate optimized results including relatively shorter processing time and lower melting temperature to guarantee that PGA becomes completely melted. As a result, at the beginning of the foaming experiment, the vessel was heated to the temperature for PGA melting (Tmelt) at 228oC which was lower than that at N2 atmosphere. CO2 was then charged into the chamber to the pressure at Tmelt (Pmelt) 2MPa which was lower than the saturation pressure (Psat) to plasticize PGA matrix. After 24


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25min in total for the equilibrium of temperature and pressure, the operational temperature was declined from Tmelt to the saturation temperature (Tsat) while the pressure was raised from Pmelt to the foaming pressure (Psat) at the same time. Within this process, scCO2 could suspend the crystallization of PGA and thereby provide a broader amorphous temperature range for foaming. At the beginning of depressurization, the so-called melt strength, elasticity and viscosity, were stronger for supporting bubble growth and stabilization in this temperature regulating window. Through such a cooling process, the PGA melt remained amorphous and was of much higher complex viscosity at 210oC, especially at low frequency, than the original one at 230oC or above. Tsat was explored with the upper threshold of 210oC to lower temperature based on the rheological results. Psat should be higher than Pmelt, as Pmelt only played the roles in providing protective atmosphere, plasticizing PGA and decreasing the melting temperature, not for the purpose of foaming. Using two stages foaming process in which the first stage used low pressure (2MPa, Pmelt) and higher melting temperature (228°C, Tmelt) and the second stage used lower saturation temperature (198-208°C, Tsat) but high pressure (10-30MPa, Psat), homogenous foams without large hollow could be produced. In addition, it was found that prolonged saturation time did not exhibit much difference from current results (data not shown).

25



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3.2 Characterization of PGA foams

Figure 3. SEM images showing the cross-sectional morphology of PGA foams prepared at different conditions. Insets are the representative SEM images of higher magnification. Bar scale: 2 µm for insets.

The cross–sectional morphology of PGA foaming samples obtained at the wide ranges of foaming temperature and pressure are shown in Figure 3. It was clear that no apparent porous structure can be observed after the foaming process performed from 198oC to 206oC under compressed CO2 of 10MPa (Figure 3a, d and g). When the foaming temperature was increased to 210oC, it seemed that bubbles underwent a process of collapse, resulting in the formation of 26


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a large cavity at the center of the sample, which should be regarded as hollows (Figure 3j). If the foaming temperature kept on increasing, the melt strength became even weaker. Correspondingly, only a PGA shell without any interior porous structures would be obtained, which agreed well with the samples foamed at higher pressure (Figure 3k and l). However, when the foaming pressure was increased to 20MPa, relatively uniform pores can be observed throughout the PGA matrix obtained at 206oC (Figure 3h), which was probably due to the enhancement of scCO2 dissolution in polymer matrix with appropriate rheological behavior. With the further increase in the pressure to 30MPa, the bubble nucleation was boosted significantly due to higher scCO2 dissolution and depressurization gradient as shown in Figure 3c, f and i. Exquisite pores were foamed due to the strong melt strength especially at 198oC, 202oC and 206oC. It is of note that the pressure of 30MPa was the upper limit of our current foaming conditions because of experimental safety and the pressure–bearing capacity of the vessel.

According to Doroudiani et al,40 in order to foam the highly crystalline polymer, the process must be carried out at a temperature above the Tm. Apparently, the current foaming strategy demonstrated the feasibility of foaming process carried out at a temperature under Tm by the plasticization effect of supercritical CO2. Due to the thermal behavior of polymer, PGA can still be completely amorphous with CO2 dissolving into the polymer melt within the cooling and second soaking process. That could be an ingenious foaming strategy for the liner– 27



Page 28 of 52

structure crystalline polymers with weak melt strength. This foaming procedure required sufficient elasticity and viscosity of polymer melt at the beginning of depressurization, otherwise the collapse of bubbles could lead to the hollows in the foams such as the PGA samples prepared at 210oC.

Figure 4. SEM images showing the effect of pressure on the morphology of PGA foams prepared at the same saturation temperature, 206oC. The associated pore size distribution (more than 200 pores counted in each operation condition), porosity (P), average diameter (D) and interconnectivity (I) were quantified by Image-Pro Plus software.

Based on the above observation and comparison, relatively homogenous pore structure 28


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with dozens of micrometers was achieved at 206oC under CO2 pressure over 20MPa. Therefore, the suitable foaming temperature should be approximately 206oC which was about 20oC lower than the melting temperature at atmospheric environment. Further efforts were made to explore the optimal foaming conditions for PGA scaffolds. Consequently, the results of PGA samples foamed at 206oC under different pressures in the range of 15MPa to 30MPa are shown in Figure 4. Detailed analysis on the pore size and distribution (more than 200 pores counted of the sample prepared in each operation condition), average pore size (D), porosity (P) and the interconnectivity (I) were given as well. It could be seen that although pores can form throughout the whole structure at 15MPa, but the walls of pores were even thicker than the size of pores. As a consequence, no statistical analysis on pore distribution was carried out for this sample. Nevertheless, for the one sample obtained at 20MPa, the average diameter, porosity and interconnectivity were 23µm, 50% and 96% respectively. When the foaming pressure was increased to 25MPa, the average diameter decreased to 21µm, while the porosity accordingly increased to 55%. Correspondingly, with the further increase in pressure to 30MPa, the average diameter reduced to less than 10µm, while the porosity increased to 60%. Meanwhile, it seemed that the pore size distribution was the narrowest for the sample obtained at 30MPa. Generally, the average diameter decreased evidently with the increase in CO2 pressure, whereas the porosity conversely increased. This was due to the fact that the nucleation and expansion of pores were motivated by the dissolved CO2, suggesting that the higher saturation pressure the higher solubility of CO2 in PGA matrix. In addition, it was noteworthy that an 29



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interconnectivity of foams over 96% could be achieved, which was one of the key issues for mass transfer of nutrients and metabolites for tissue engineering.

Figure 5. SEM images showing the effect of temperature on morphology of PGA foams fabricated at the same pressure, 20MPa. The associated pore size distribution (more than 200 pores counted in each operation condition), porosity (P), average diameter (D) and interconnectivity (I) were quantified by Image-Pro Plus software.

Notably, when the foaming pressure was set up to 25MPa and further on, the average diameter of foams might have been too small for tissue engineering. Hence, the following efforts returned to focusing on the investigation of fine temperature effect near 206oC under the fixed pressure of 20MPa. The corresponding results are shown in Figure 5. Under the foaming pressure of 20MPa, the average diameter increased from 14µm to 38µm and the porosity 30


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increased from 45% to 74%, corresponding to the foaming temperature from 204oC to 208oC. However, the pore size distribution was clearly further broadened. Importantly, the interconnectivity was over 92% for the foamed PGA.

Generally, pore size increases with lower depressurization rate, hence some depressurization duration in solid-state foaming can even be 120 min to achieve lower depressurization rate and larger pores.41 However, not only the dissolution or escape rate of CO2 but also heat transfer and polymer properties affect the pore properties. For melt-state foaming, the depressurization duration is usually much shorter, e.g. 0.2 second.28 Uniquely, we discover that depressurization rate was not a dominated factor for pore property in PGA foaming. Whilst the temperature at saturation stage was more crucial for the pore property. PGA had low melt strength and was very sensitive to the processing temperature. Merely a 2oC temperature difference led to completely different foaming morphology.

In fact, formation of pores in the foaming process usually comprise of three stages: nucleation, bubble growth, and stabilization. In batch foaming, the shear force during bubble growth is much weaker compared to that in the extrusion or the injection molding processes. The melt strength of polymer melt matrix has different impacts on the bubble growth and stabilization stages. It forms resistance to the bubble growth, whereas is important in supporting the foamed pores from collapsing during stabilization.42-43 Usually, the melt strength and the kinematic viscosity of polymer melt decrease rapidly with the increase in 31



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temperature. For PGA melts, the elasticity as well as complex viscosity as shown in Figure 1, within the low frequency region, exhibited great dependence on testing temperature. In this work, the foaming results of PGA melt showed obvious dependence on foaming temperature, which meant that the batch foaming procedure for PGA conformed to the rheological properties under low frequency. Higher elasticity and viscosity of PGA melt at lower Tsat with low shear rates at the beginning of depressurization could thus permit a controlled pore growing manner and was advantageous to the quicker fixation of bubbles. 3.3 Nano–scale morphology on bubble surface

Figure 6. Schematic illustration of the generation of nano-features on foamed PGA scaffolds.

Under close magnification, the nano–scale textures on foamed PGA bubble surface were discovered as shown in Figures 4 and 5. The representative structure was further enlarged in 32


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the red box in Figure 6. These features may be caused by the aggregate states and the tensile force during the bubble growth and shaping process as illustrated in Figure 6. There was a difference between the density of amorphous state, 1.45–1.50g/cm3 and that of crystal state, 1.69–1.71g/cm3, for PGA 34, which was also possible in the present supercritical fluid assisted foaming technology. PGA matrix can shrink from melt to crystals during the bubble growth and stabilization process with the decrease in temperature. In this process, the nucleation sites of crystals were produced in the solid part, bubble walls of foams, because of the rapid PGA crystallization rate and the bubble growing tensile force. The tensile forces on such sites in planar plan were from all sides since the crystal was several magnitudes smaller than the whole bubble. As long as the crystalline site was in the point of force equilibrium, a two–dimensional spherocrystal will be formed. At the edge of a bubble, the tensile force on one direction could be larger than that of other orientations. Under this circumstance, the spherocrystal growth will be interrupted and stretched to be a zonal structure. The growth of crystals could be regarded as a two dimensional process due to the thin bubble wall. Limited crystals can grow into a planar disk. It should be noted that the density change induced by crystallization was a retractile behavior opposite to the tensile force. Fibrous structure is produced in such an inverse force field. Additionally, the thickness of several peaks was measured in the range of 21–26nm, which was quite close to the crystal size of 18.6nm determined by the Scherrer formula.

It was interesting to notice that nano–sized morphology features were observed on the 33



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internal surface of PGA foams, since it was usually accepted that the surface morphology of polymeric biomaterials is one of the major issues influencing the interconnection between the scaffolds and tissue cells in regenerative medicine.44 Cell shape can even be programmed by the nano–structures on the surface of the films to affect stem cell differentiation.45 Furthermore, the influence of hierarchical pores on bone regeneration, was proven to be essential in mineralization and mechanical improvement during tissue regeneration.46-47 Therefore, it was proposed that the current nano–sized surface features could be beneficial for the subsequent cellular behaviors, which would be an interesting point that remains to be investigated in detail. 3.4 Mechanical properties

Figure 7. (a) A typical compression load-displacement curve of PGA foams prepared at 208oC and 20MPa. (b) XRD patterns of the unprocessed PGA particles and PGA foams. The PGA foams were prepared at 208oC and 20MPa.

Table 1. Compressive Modulus and crystallinity of selected PGA foams 34


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Sample

Foaming T/

Foaming P/

Compressive Modulus/

C

MPa

MPa

1

206

20

90.1±9.1

53 ± 3.0

2

206

25

98.0±11

51 ± 2.1

3

206

30

116.2±5.0

53 ± 2.9

4

204

20

105.9±10.3

51 ± 1.8

5

208

20

68.7±6.1

46 ± 1.1

o

Crystallinity/ %

The mechanical properties of the obtained PGA foams with good morphology were characterized by compressive tests. The typical compressive load–displacement curve of porous foam fabricated at 208oC and 20MPa is shown in Figure 7a, and the corresponding compressive modulus of foamed PGA samples are listed in Table 1. As seen in Figure 7a, the PGA foam exhibited only the linear elastic deformation when subjecting to compression load up to 500N without the appearing stress plateau and densification region in their compressive patterns. According to Gibson et al,48 the compressive stress–strain curves of porous foams can be divided into three parts: the initial linear elastic region dominated by pore wall bending and the counterforce of compressed air, a stress plateau caused by pore collapse, and a stress increasing region (densification region) where the pores have completely collapsed and the load is applied to a bulk–like material.

Based on the values in Table 1, all of the compressive modulus of the obtained PGA foams were over 65MPa. Such a modulus approximated to that of cancellous bone, 71MPa.49 The excellent mechanical properties can be inferred to the rapid crystallization of PGA induced by compressed CO2. It was reported that semi–rigid chains connecting 35



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crystalline domains were responsible for transferring the stress between crystalline domains and undergo tensile deformation.50 For the PGA foams under compression in this work, the pileup of PGA crystals in the entity region could also contribute to the resistance force with the semi–rigid part transferring that stress. Derived from the load-displacement curves, it was found that even sample 5 which had lower crystallinity could resist 500N without collapse. It may be because that the pore structure and property in current PGA porous scaffolds were different from these formed by routin techniques, e.g. salt leaching-solvent evapoartion. The smaller pores in current PGA scaffolds were developed from single pores. Numerous smaller pores, 10-40µm, remained around larger pores. The larger pores came from the coalescence of smaller pores, evidenced by the unique pore morphologies that the thick and thin rods were across larger pores as seen in Figure 8. Parts of the arched structure of smaller pores were reserved to transfer pressure.

Figure 8. A typical SEM image of PGA foams prepared at 208oC and 20MPa showing the 36


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reserved small pores within and around large pores (indicated by blue and red frames) and the thick and thin rods across larger pores (indicated by yellow frames).

Since PGA possesses excellent biocompatibility, processing into porous structure with great compression capacity by this study will fill a technical gap, which is especially beneficial for bone tissue engineering. In this work, PGA foams with strong mechanical properties can be produced with only a simple temperature regulation procedure without any concerns for the possible toxicity of the organic solution.

The crystallization status of unprocessed PGA granules and foamed PGA matrix were both measured by XRD as shown in Figure 7b. An excellent consistence in XRD patterns of different PGA samples was displayed, indicating that crystals also formed during the bubble growth process of foaming. A nonlinear crystalline result for the foamed PGA samples was obtained by the DSC test, and the crystallinity of different samples were in the region of 50% as shown in Table 1, confirming the above demonstrated excellent mechanical properties. Considering the morphology and mechanical properties of PGA foams, the one prepared at 208oC and 20MPa, was further used for the following in vitro and in vivo cellular experiments.

37



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3.5 Cell distribution and viability

Figure 9. SEM and confocal images of human fibroblasts cultured on PGA scaffolds for 1 and 3 days. After being seeded on the scaffold for 1day, fibroblasts attached tightly on the scaffold with prominent cytoplasmic processes according to SEM (Figure 9a, c and e) and confocal images (g and i). After 3 days of culture, abundant production of ECM can be visualized, covering the pores of scaffold according to SEM (b, d and f) and confocal images (h and j).

SEM was carried out for the direct visualization of fibroblast morphology and ECM deposition on PGA scaffolds after fibroblasts were cultured for 1 and 3 days. As shown in Figure 9a–f, after 1 day, the fibroblasts anchored themselves tightly onto the surface of scaffolds and displayed an evident cytoplasmic process. Cell–cell communication was already established since the cell–cell contact can be already observed. When the culture time was extended to 3 days, abundant production of ECM was found to cover the pores of these scaffolds. Cell layers were formed on the surface of pores, and the scaffolds were almost entirely surrounded with cellular layers. The above results indicated the outstanding 38


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biocompatibility of PGA foams in such a porous form to support the initial attachment and subsequent proliferation of fibroblasts in vitro.

The distribution and growth of fibroblasts at 1 and 3 days after being seeded on the scaffolds was also visualized by confocal images as shown in Figure 9g– j. Vivid cytoplasmic extension and cellular communication had been established after 1 day. A similar trend involving cell proliferation was markedly observed, this was confirmed by SEM when the culture time was increased from 1 day to 3 days. 3.6 Proliferation of fibroblasts on the scaffolds

4

25 Cell Number/ 1×10

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

Control Experimental

15 10 5 0 1d

3d

7d

Figure 10. Proliferation of fibroblasts on PGA scaffolds with time by CCK8 assay. PGA scaffolds of 7×7×1 mm, prepared at 208 oC and 20 MPa were served as the experimental group, while petri dishes (Φ 15.6 mm) were served as the control group to evaluate the proliferation of fibroblasts on them. All values are the means ± SD; n= 3. * indicates significant difference between the control and experimental groups (p

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