Antimicrobial properties and osteogenicity of vancomycin-loaded

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Antimicrobial properties and osteogenicity of vancomycinloaded synthetic scaffolds obtained by supercritical foaming C. A. Garcia-Gonzalez, Joana Barros, Ana Rey-Rico, Pablo Redondo, José L GómezAmoza, Angel Concheiro, Carmen Alvarez-Lorenzo, and Fernando J Monteiro ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17375 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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ACS Applied Materials & Interfaces

Antimicrobial properties and osteogenicity of vancomycin-loaded synthetic scaffolds obtained by supercritical foaming Carlos A. García-Gonzáleza,*, Joana Barrosb, Ana Rey-Ricoc, Pablo Redondoa, José L. GómezAmozaa, Angel Concheiroa, Carmen Alvarez-Lorenzoa, Fernando J. Monteirob a

Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, R+D Pharma group (GI-1645), Facultad de Farmacia and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, E-15782-Santiago de Compostela, Spain b

FEUP-Faculdade de Engenharia, Universidade do Porto, I3S-Instituto de Investigação e Inovação em Saúde, and INEB-Instituto de Engenharia Biomédica, Porto, Portugal c

Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg, Germany

*

Corresponding author: [email protected]; phone: +34 881 815252; fax: +34 981 547148.

Keywords: polymeric scaffold; vancomycin; supercritical CO2; foaming; bone regeneration.

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Abstract Advanced porous synthetic scaffolds are particularly suitable for regeneration of damaged tissues, but there is the risk of infections due to the colonization of microorganisms forming biofilms. Supercritical foaming is an attractive processing method to prepare bone scaffolds regulating simulataneously the porosity and loading of bioactive compounds without loss of activity. In this work, scaffolds made of poly-ε-caprolactone (50 kDa) containing chitosan and an antimicrobial agent (vancomycin) were processed by supercritical CO2 foaming for bone regeneration purposes. Obtained scaffolds showed a suitable combination of morphological (porosity, pore size distribution, interconnectivity), time-dependent in vitro vancomycin release behaviour and biological properties (cell viability and proliferation, osteodifferentiation and tissue-scaffold integration). Scaffolds sustained vancomycin release for more than two weeks. Finally, the antimicrobial activity of the scaffolds was tested against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria after 24 hours incubation with full growth inhibition for S. aureus.


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Abbreviations: ALP, alkaline phosphatase ANOVA, analysis of variance ATCC, American type culture collection CAM, chorioallantoic membrane CFUs, Colony Forming Units Chit, chitosan DMEM, Dulbecco's Modified Eagle's Medium DSC, differential scanning calorimetry FBS, fetal bovine serum LDH, lactate dehydrogenase MIC, minimum inhibitory concentration MIP, mercury intrusion porosimetry MRSA, methicillin-resistant Staphylococcus aureus MSCs, mesenchymal stem cells PBS, phosphate buffer solution PCA, plate count agar PCL, poly(ɛ-caprolactone) scCO2, supercritical carbon dioxide sc-drying, supercritical drying SEM, scanning electron microscopy SS, absolute sum of squares Tm, melting temperature TSB, tryptic soy broth V, vancomycin


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1. Introduction Synthetic scaffolds represent the most beneficious strategy to get the best performance-toavailability ratio for bone repair in the regenerative medicine discipline. Scaffolds are endowed with morphological properties as well as biological cues able to promote firstly cell colonization and then tissue growth. Nevertheless, scaffolds as indwelling devices originate biomaterialbiological tissue interfaces able to be colonized not only by human cells but also by bacteria forming biofilms 1. The risk of infection after surgical implantation is estimated at ca. 1-2%, represents half of the intrahospitalary infections and its treatment generates high socio-sanitary costs (ca. 25,000 € per patient)2-3. Infections cause serious complications implying from scaffold replacement up to compromising the life of the patient 1. Despite important progresses in aseptic and prophylactic practices during the surgical procedures, the prevalence of infections during the post-implantation time period is still unacceptable. Particularly, the systemic prophylaxis using antibiotics has limited success because of the poor irrigation of the affected tissue and the diffusional constraints imposed by the intricate porous geometry of scaffolds to the access of the drug 3. The conferral of antimicrobial properties to the bone porous scaffolds is regarded as an auspicious solution to prevent post-surgery infectious events. The use of antibacterial coatings and the incorporation of antimicrobial agents in the scaffold formulations are among the most promising strategies to prevent the bacterial colonization of the scaffolds

4-5

. In the latter case,

the local delivery of the antimicrobial agent would reduce the administered dose as well as mitigate resistance-inducing and other side effects of the drug when compared to systemic delivery by intravenous infusion, the most common current clinical practice 3. However, the selected antimicrobial agent to be incorporated in the scaffolds should be bioactive against a broad spectrum of bacteria 6, its release should be modulated to preserve its local biological effect over a sufficient post-surgical time period (in the order of days to weeks)7 and should not hamper the colonization of the grafts by the host cells 1. The local and sustained release of antimicrobial agents is thus regarded as a safe therapy preventing the development of bacterial resistance 7.


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Vancomycin is a tricyclic glycopeptide with a relevant antibiotic activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA), the most common pathogen from hospitalary infections 8. Its mechanism of action is the inhibition of the peptidoglycan synthetic pathway in the bacterial cell wall

9

. Vancomycin has limited

antimicrobial activity against Gram-negative bacteria due to the molecular size-exclusion effect of the bacterial outer membrane. Nevertheless, vancomycin is effective against Gram-negative bacteria from certain strains (Neisseria species)10, or when synergistically combined with other antibiotics under subinhibitory concentrations (e.g., trimethoprim and nitrofurantoin)

11-12

.

Moreover, vancomycin presents a dose-dependent toxicity, being safe over a wide dose range for bone cells, and does not compromise the osteoblastic differentiation of mesenchymal stem cells (MSCs) 13-15. The incorporation of antibiotics like glycopeptides (e.g., vancomycin) or β-lactams (e.g., cefuroxime) in the scaffolds is severely limited by the low thermal stability of these compounds 16-17

. Thermally-intensive processing of scaffolds (e.g., hot melt molding, extrusion, thermally

induced phase separation, conventional foaming) is thus precluded. Solvent-based processing methods (e.g., solvent casting, particulate leaching, electrospinning) usually require the use of organic solvents, which are cytotoxic, and of rigorous downstream processes of purification resulting in cumbersome protocols, and they lead to low antibiotic incorporation yields due to intensive leaching. Supercritical CO2-assisted foaming emerges as the most robust alternative for the solvent-free processing of scaffolds under temperatures below their normal melting points

18-19

. This technique has been already tested with other thermally sensitive active agents

like growth factors with incorporation yields and retained activities close to 100% 20-21. In this work, poly(ɛ-caprolactone) (PCL) scaffolds loaded with different vancomycin contents (0 to 7 wt.%) were processed by supercritical foaming. The effect of the presence of chitosan, a semisynthetic polysaccharide with certain cell interaction and differentiation properties well as antimicrobial properties against Gram-positive and Gram-negative bacteria

23

22

as

, in the

scaffold composition was evaluated. The obtained scaffolds were tested regarding morphological properties (porosity, density, roughness, interconnectivity) and their infiltration


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capacity with bone marrow-derived mesenchymal stem cells (MSCs). The scaffolds were challenged against S. aureus (Gram(+)), the prevailing virulent bacteria for early infections, and E. coli (Gram(-)), a common bacterial source of intrahospitalary infections. The viability and proliferation of MSCs and osteoblasts in the presence of the vancomycin-loaded scaffolds as well as the differentiation of MSCs towards osteoblasts were tested. Finally, in ovo tests were undergone to evaluate the biological tissue-scaffold integration. 2. Materials and methods 2.1. Reagents PCL (PCLraw, 50 kDa, Tm=61.7 ºC, crystallinity: 59.0%) in the powdered form was purchased from Polysciences (Warrington, PA, USA). Vancomycin (V, 1063 IU/mg) and chitosan (Chit, deacetylation degree: 77.6-82.5%; mean particle size: 79.0 µm; nominal viscosity at 1 wt. % in 1% acetic acid and at 20 °C: 71-150 mPa·s) were provided by Fagron Ibérica (Terrassa, Spain) and Chitoscience (Saale, Germany), respectively. Carbon dioxide (99.9% purity) was supplied by Praxair, Inc. (Madrid, Spaini). Sodium hydroxide (99% purity, VWR International, Geldenaaksebaan, Belgium) and potassium dihydrogen phosphate (99% purity, Panreac Química, Barcelona, Spain) were used to prepare the PBS pH 7.4 buffer solution. For the cell studies, DMEM culture medium, phosphate buffered saline (Dulbecco's Phosphate Buffered Saline) and penicillin/streptomycin solution were from Sigma. Fetal bovine serum (FBS) was from Gibco; LDH (Cytotoxicity Detection Kit LDH) and WST-1 (Cell Proliferation Reagent WST-1) were from Roche™; Quant-iT Proliferation Kit PicoGreen dsDNA was from Molecular Probes Inc. 2.2. Porous PCL-vancomycin composites scaffolds preparation Powdered mixtures (1.8 g) with different PCL, vancomycin (V) and chitosan (Chit) contents were weighed, introduced into cylindrical Teflon moulds, physically mixed and compacted. Samples were placed in a high pressure stainless steel vessel (Thar Technologies, Pittsburgh, PA, USA) and pressurized with CO2 under supercritical conditions (40°C and 140 bar) and under agitation (700 rpm) for 1 h. After the so-called soaking period, the vessel was depressurised until atmospheric pressure at a CO2 flow rate of 1.8 g/min. Finally, the obtained


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scaffolds were stored overnight in closed containers and in darkness and then their weight and dimensions measured. The external surface of the scaffolds was surrounded by a non-porous layer of 10-20 μm thickness, typical from the supercritical foaming processing

18, 24

, and was

removed using a scalpel. The resulting scaffolds were denoted as PCL-xV-yChit, where x and y correspond to the initial weight content (in percentage) of vancomycin and chitosan in the powdered mixture, respectively (Table 1). 2.3. Structural and physicochemical characterization of scaffolds Scaffolds were characterized by digital imaging using a CCD Microscope Camera (DFC7000T, Leica, Wetzlar, Germany) operating at room temperature. Moreover, micrographs of the scaffolds were also recorded by scanning electron microscopy (SEM, EVO LS15, Zeiss, Oberkochen, Germany). Scaffolds were previously cut with a surgical blade in the form of discs and the same scaffold analyzed with both imaging techniques without any further pretreatment. Scaffolds were studied regarding their textural properties using mercury-intrusion porosimetry (MIP) analysis (Micromeritics Autopore IV 9500, Norcross, GA, USA). Pore size distributions, total pore volumes (Vp,MIP), specific surface areas (AMIP), mean pore diameters (dp,MIP) and pore median were accordingly determined. Thereafter, a 3D network model of the macroporous structure from the MIP-cumulative curves of scaffolds was obtained using PoreXpert v.1.6.567 software (PoreXpert Ltd, Plymouth, United Kingdom)

25

. Pore interconnectivity and water

permeability (1.03 bar, 25 ºC) were calculated using this model. The filtration module from the same software was used to obtain the infiltration capacity of MSCs in the scaffolds by simulating the flow of the MSCs (23.9±5.2 µm diameter, an average value for human MSCs from bone marrow25) from the top to the bottom of the unit cell. The fractal dimensions of the inner porous surface of the scaffolds were obtained from the pore size distribution data using Eq. (1) 26

⁄ = ∙ +

Eq. (1)

where Wn is the accumulated surface energy in the mercury intrusion process up to the nth stage, C is calculated from the surface tension (47.5 N·m-2) multiplied by the cosine of the mercury


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contact angle (ca. 130º). Vn and rn are the pore volume and the pore radius at the nth stage of mercury intrusion process, respectively, and Ds denotes the fractal dimension of the inner porous surface. Melting behavior and crystallinity of neat PCL and PCL-processed scaffolds were evaluated from DSC analysis (DSC-Q100, TA Instruments; New Castle, DE, USA). The materials were heated up to 250 °C at a rate of 10 °C/min under inert atmosphere (N2, 50 mL/min). Melting temperature (Tm), heat of melting (∆Hm) and percentage of crystallinity (assuming a heat of fusion for the perfect PCL crystal of 142 J/g 27) of PCL in the scaffolds were obtained from the DSC-thermograms. The skeletal density (ρskel) of the raw materials (V, Chit and PCL) and the prepared scaffolds were measured at 25 ºC and 1.03 bar using a helium-pycnometer (Quantachrome; Boynton Beach, FL, USA). Values were determined from five replicates. The bulk density (ρbulk) of the cylindrical scaffolds obtained by supercritical foaming was determined from the dimensions and weight of the samples after the foaming process (in triplicate). The resulting overall porosity (ε) of the scaffolds was calculated following Eq. (2). = 1 −

! ∙ 100

(2)

2.4. Vancomycin release studies Scaffold pieces (100 mg) were suspended in glass flasks containing 25 mL of PBS pH 7.4 medium. The dissolution profile in 100 mL of PBS of 50 mg of pure vancomycin powder was also performed as reference for the release profiles. Release studies were carried out at 37ºC and 60 rpm for 2 weeks using a thermostatic oscillating bath (Unitronic 320 OR, JP Selecta, Barcelona, Spain). Aliquots of 3 mL were sampled through that period at selected times. Withdrawn volumes were replaced with fresh medium. Vancomycin concentration was measured by UV-Vis spectrophotometry (8453 model, Agilent Technologies, Santa Clara, CA, USA) at 281 nm. Vancomycin standard solutions were performed in PBS at concentrations ranging from 2.5 to 100 µg/mL and in triplicate (R2>0.99). Vancomycin release studies were performed in triplicate and the obtained drug concentrations in the release medium were always


8

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ten times lower than its maximum drug solubility (i.e. sink conditions) to avoid saturation. Modeling of the vancomycin release from the scaffolds was carried out by fitting the obtained release data to a biexponential equation (3) corresponding to two simultaneous first-order dissolution processes (GraphPad Prism version 6.04 for Windows -GraphPad Software, La Jolla, CA, USA- software). = #$%,' (1 − ) *+ , - + #$%, (1 − ) *+. , -

(3)

where D denotes the dosage percentage of vancomycin released at time t, in %; Dmax,i, the maximum dosage percentage of vancomycin released in stage i, in %; and ki, the release rate coefficient in stage i, in h-1. 2.5. Antimicrobial activity The reference strains S. aureus ATCC 25923 and E. coli ATCC 25922 were used. For the quantification of sessile bacteria on vancomycin-loaded PCL scaffolds and planktonic bacteria in the culture medium, a bacterial suspension of concentration 1.0·108 cells/mL in Tryptic Soy Broth (TSB) for 24 h at 37 °C was prepared from each strain. Then, the scaffolds (nine replicates), in the form of discs (10 mm diameter, 1 mm length) and previously UV-sterilized, were placed in the wells of 48-well plates (Cellstar, Greiner Bio-One, Monroe, NC, USA) containing 1 mL of the bacterial suspension and incubated at 37º C for 24 h. For sessile bacteria quantification, the scaffolds (nine replicates) were carefully withdrawn after the incubation and washed with sterile 0.9 % (w/w) NaCl aqueous solution to remove loosely attached bacteria. Then, six scaffold replicates were transferred to Falcon tubes containing 3 mL of 0.9 % (w/w) NaCl solution and sonicated for 20 min in an ultrasonic bath (Bandelin Sonorex Digitec, Berlin, Germany) to detach the sessile bacteria. Then, the suspension was serially diluted and the Colony Forming Units (CFUs) were estimated by spread plating in Plate Count Agar (PCA). The plates were incubated 24 h at 37 ºC. Results were expressed as the decimal logarithm of the number of attached bacteria per milliliter of suspension. All tests were performed in triplicate. The sessile bacteria on vancomycin-loaded PCL scaffolds were directly observed by SEM microscopy from the other three scaffold replicates. Scaffolds were fixed in 3% (v/v)


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glutaraldehyde (Fluka, Germany) at room temperature for 30 min, and then gradually dehydrated in a gradient series for 10 min each step. Scaffolds with attached S. aureus bacteria were dehydrated in ethanol (50, 70, 90 and 100%), and those with attached E. coli bacteria were dehydrated in hexamethyldisilazane (HMDS, Sigma-Aldrich) (50, 70, 90 and 100%). Then, scaffolds replicates were fixed on sample holders with double-sided carbon tape and sputtercoated (SPI-Module) with a conductive gold–palladium film. Biofilms were imaged using a FEI Quanta 400 FEG/ESEM microscope (FEI, Hillsboro, OR, USA) operated at 15 kV. SEM with magnifications were obtained between 200 and 2,000x. The planktonic bacteria susceptibility to vancomycin-loaded PCL scaffolds was quantified by optical density using a UV/Vis spectrophotometry (Synergy Mx plate reader, BioTek Instruments, Winooski, VT, USA) at the absorbance of 640 nm. 2.6. Cell seeding and culture Bone marrow aspirates were obtained from the distal femur of patients undergoing total knee arthroplasty and MSCs were isolated and expanded in culture using standard protocols 28. Bone samples were retrieved from osteoarthritic joints of patients and osteoblasts were isolated by enzymatic digestion of bone explants as previously described 29. All patients provided informed consent before being included in the study and procedures were in accordance with the ethical principles of the Helsinki Declaration. Cells (hMSCs passage 4-5 and osteoblasts passage 2-3) were maintained in DMEM, 10% FBS, 100 U/mL penicillin G, 100 µL/mL streptomycin (growth medium) and used for the different experiments. For cytotoxicity and cell proliferation assays cells were seeded in 24-well plates (20,000 cells/well) and incubated at 37ºC during 12 h before adding the scaffolds. For cell attachment and ALP assays, scaffolds were placed in 48well plates and a density of 10,000 cells per scaffold was involved. 2.7. Cytotoxicity and cell proliferation tests Viability of MSCs or osteoblasts upon contact with the scaffolds was quantified in duplicate at 24 hours using the Cytotoxicity Detection KittPLUS (LDH) to measure the release of lactate dehydrogenase (LDH) activity in the supernatants of culture medium 30. Absorbance at 490 nm


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was measured using a microplate reader (BIORAD Model 680, Hercules, CA USA) and the percentage of cytotoxicity was calculated as: /0121234/410 % = 7)38)49)1:; − ? × 100

(4)

where low control (LC) corresponds to cells without assay treatment and high control (HC) to cells placed in the lysis buffer provided in the kit. Viability was also determined in duplicate using the Cell Proliferation Reagent WST-1 where the absorbance values are proportional to the cell numbers

28

. Cells maintained in culture

medium but in absence of the scaffolds were used as negative control. The % of cell survival was calculated as follows: viability (%) = [absorbance of the sample/absorbance of the negative control] x 100(5) Proliferation of the MSCs seeded on the scaffolds was monitored in triplicate after 3 and 12 days by measuring the DNA content using a commercial PicoGreen kit (Quant-iTPicoGreen dsDNA, Invitrogen, Molecular Probes Inc., Eugene, OR, USA). Briefly, scaffolds were washed with PBS followed by three freeze-thaw cycles and then by sonication before DNA analysis. Cell lysates (50 µL) were mixed in equal parts with aliquots form working solution (prepared following the instructions from the manufacturer) and incubated for 5 min protected from the light. DNA contents were measured in a fluorescence plate reader (BMG LabTech GmbH, Ortenberg, Germany) at the wavelengths of λexc = 485 nm and λem = 520 nm by using a DNA standard curve 31. 2.8. Cell attachment tests After 7 days of culture, MSCs-seeded scaffolds were washed with PBS and processed for microscopy in duplicate. Cell viability on the scaffolds was monitored by using a LIVE/DEAD assay kit following the instructions from the manufacturer. Briefly scaffolds were stained with 150 µL of LIVE/DEAD reagent (2mM calcein-AM and 4mM ethidium homodimer in PBS) and incubated for 20 min in darkness. Imaging from the scaffold was carried out using a LEICA MZ16F fluorescence microscope (LEICA Microsystems Heidelberg GmbH, Mannheim, Germany).


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To evaluate the spreading of MSCs onto the scaffolds, constructs were stained with phalloidin/DAPI to visualize the cellular actin-cytoskeletal and nucleus, respectively. Shortly, MSCs-seeded scaffolds were fixed with paraformaldehyde (4%) for 10 min and washed three times with PBS. After washing, specimens were permeabilized by incubation in 0.2% Triton/PBS solution pH 7 for 5 min, rinsed again and incubated in darkness for 20 min with a solution of BODIPY 650/665 Phalloidin reagent (Molecular Probes Inc., Eugene, OR, USA) in PBS. Finally, scaffolds were washed with PBS, placed on glass slides, and one drop of the DAPI-containing ProLong® Gold Antifade Mountant (Molecular Probes Inc., Eugene, OR, USA) was added to each scaffold. Cell-seeded constructs were stored at -20ºC until visualization with a Leica TCS-SP2 spectral confocal microscope (Leica TCS-SP5, LEICA Microsystems Heidelberg GmbH, Mannheim, Germany). 2.9. ALP activity ALP activity was used in MSCs-seeded scaffolds as an early marker of osteogenic differentiation at 3 and 12 days post-seeding 31. Scaffolds (in triplicate) were washed with PBS and MilliQ water and then subjected to three freeze-thaw cycles to lyse the cells. Aliquots from cell lysates (50 µL) were incubated with 150 µL of ALP substrate (p-nitrophenol phosphateliquid substrate system) in 96-well plates at 37ºC for 30 min. Absorbance at 440 nm was measured using a Fluostar Optima fluorescence plate reader (BMG LabTech GmbH,Ortenberg, Germany). A p-nitrophenylphosphate calibration curve (R2>0.99) was used to determine the ALP concentration of the samples. The results from ALP activities (in nmol/min) were normalized by the amounts of DNA quantified (see cell proliferation in Section 2.7). 2.10. in ovo tests CAM integration test was performed according to a previously reported protocol

25, 32

with

fertilized hen’s eggs (50-60 g) obtained from Coren (Ourense, Spain). Briefly, after incubation at 37°C and 60% relative humidity for 10 days, a cut (1 x 1.5 cm) was practiced on the longitudinal side of the egg to remove the shell and then cylindrical pieces (d=4.9 mm; h=1.9 mm) from the scaffolds (n=3) were located on the CAM of the eggs. The windows were sealed


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with parafilm and the eggs incubated for 7 more days. Then, the scaffolds were collected, the outer integrated tissue removed with a scalpel and the inner tissue was fixed in paraformaldehyde solution (4 % (w/v), 10 min). The integration of the scaffolds to the CAM was inspected by confocal microscopy using a DAPI/phalloidin staining (cf. Section 2.8). 2.11. Statistical analysis All results were expressed as mean ± standard deviation. Statistical analyses of antimicrobial experiments, cell viability, cell proliferation and ALP activity were performed using ANOVA tests (GraphPad Prism 6, GraphPad Software, Inc., La Jolla, CA, USA) followed by the posthoc Tukey HSD multiple comparison test was used to determine the significant difference (p). 3. Results and Discussion 3.1 Scaffolds development and morphological characterization Processing of scaffolds using scCO2 foaming exploits the plasticizing effect of CO2 on thermoplastic polymers

18, 33

. Accordingly, the melting point of PCL decreased as a function of

the CO2 content, being the depletion effect dependent on the operating temperature and pressure 33-35

. In this work, the melting point of the used PCL decreased from its normal melting point

(61.7ºC) to a value below the supercritical foaming operating temperature (40ºC) with CO2 pressures above 89.3±0.9 bar according to view cell measurements (Fig. S-1). The operating pressure used determines the amount of CO2 that is absorbed in the PCL-based matrix during the soaking period. This value is closely related to the extent of the plasticizing effect of the fluid and increases with higher pressures 24. Foaming experiments were accordingly carried out at 40ºC and 140 bar during 60 min to ensure the complete melting of the PCL and a high CO2 sorption in the polymeric matrix. Upon CO2 removal from the molten polymeric matrix (i.e. depressurization), pores are formed through a nucleation-growth mechanism taking place in parallel to an increasing vitrification of the PCL as CO2 pressure decreases. A slow depressurization rate (1.8 g/min) at 40ºC was chosen in this work to promote diffusion of CO2 from the polymer to the fluid bubbles thus favoring the growth of the pores. The end result is a solid porous material with high porosity (ca. 70%) and large macropores with smooth pore surface and interconnected porosity (Table 1 and Figs. 1a and 2a) 19-20.


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The feasible mild operating temperature coupled to the absence of organic solvents when using the supercritical foaming technique opens up a new processing window for scaffolds containing thermally-sensitive bioactive compounds with retained activity and high incorporation yields (ca. 100%)

19-21, 36

. These features of supercritical foaming are relevant if compared to other

scaffolds manufacturing approaches and match the specific processing demands of vancomycin, a bioactive agent showing decreased stability and antimicrobial activity under mild temperature and when dissolved in certain solvents 37-39. The incorporation of vancomycin to the PCL-based scaffold had not a significant effect on the overall porosity (ε) even at high drug contents (5 wt.%) (Table 1), although a limited reduction in the mean pore size (dp,MIP) was obtained with increasing vancomycin content (Fig. 1). Scaffolds showed homogeneous distribution of the components in the material, except for the PCL-7V-0Chit scaffolds where the high vancomycin content (7 wt.%) resulted in segregation of the components (Fig. 1d). The incorporation of chitosan in the scaffolds formulation resulted in a decreased porosity (Fig. 3). The dependence of porosity on chitosan content can be ascribed to the non-foamability of chitosan by scCO2 resulting in an increased bulk density (ρbulk) (Table 1). Scaffolds with 5-10 wt.% chitosan had smaller pores and higher pore interconnectivity with broader pore throats (Fig. 3b,c). Higher chitosan contents (15 wt.%) resulted in a loss of mechanical integrity and heterogeneous composition with phase segregation (Fig. 3d). Finally, the presence of vancomycin and chitosan in the scaffolds provided a certain roughness to the pore surfaces (Fig. 2b-d). A rough microstructure on the pores of the scaffolds usually results in enhanced solidliquid interfaces with respect to smoother ones and promotes a uniform cell attachment and proliferation 18, 40.


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Fig. 1: Optical micrographs of PCL-based scaffolds obtained by supercritical foaming and with increasing vancomycin content: (a) PCL-0V-0Chit, (b) PCL-1V-0Chit, (c) PCL-3V-0Chit and (d) PCL-7V-0Chit. Scale bar: 1mm. High vancomycin content resulted in segregation of the components as observed for PCL-7V-0Chit scaffold (white arrows).


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Fig. 2: SEM pictures of the porous surface of PCL-based scaffolds obtained by supercritical foaming: (a) PCL-0V-0Chit, (b) PCL-0V-10Chit, (c) PCL-1V-0Chit and (d) PCL-5V-10Chit. Scale bar: 100 µm. The presence of chitosan and vancomycin provided surface roughness to the inner pores.


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Fig. 3: Optical micrographs showing the effect of increasing chitosan content in the scaffolds on the porous structure: (a) PCL-5V-0Chit, (b) PCL-5V-5Chit, (c) PCL-5V-10Chit and (d) PCL5V-15Chit. Scale bar: 1 mm. High chitosan contents (15 wt.% and higher) resulted in scaffolds with severe heterogeneity in the composition (asterisk points at a chitosan-rich region of the scaffold). Table 1: Composition, morphological and textural properties of supercritically processed scaffolds. Values expressed as mean values and, in parenthesis, standard deviation (n=3). ρbulk, Scaffold

3

g/cm

ρskel, 3

g/cm

ε,

AMIP,

%

2

m /g

Vp,MIP, 3

cm /g

dp,MIP,

Pore

µm

median, µm

PCL-0V-0Chit PCL-0V-10Chit PCL-1V-0Chit PCL-3V-0Chit PCL-5V-0Chit PCL-5V-5Chit PCL-5V-10Chit PCL-5V-15Chit

0.338 (0.001) 0.493 (0.019) 0.369 (0.001) 0.358 (0.002) 0.376 (0.002) 0.372 (0.018) 0.402 (0.012) 0.401 (0.009)

1.109 (0.006) 1.146 (0.010) 1.125 (0.004) 1.115 (0.007) 1.120 (0.006) 1.111 (0.016) 1.118 (0.021) 1.134 (0.006)

69.5 (0.1) 57.1 (1.7) 69.3 (2.2) 69.2 (0.5) 68.5 (0.9) 66.5 (1.6) 64.1 (1.1) 64.7 (0.8)

5.66

0.32

0.23

28.8

6.10

0.54

0.35

80.2

6.04

0.41

0.27

49.2

6.55

0.32

0.19

19.7

6.19

0.28

0.18

52.3

7.01

0.30

0.17

46.4

6.67

0.17

0.10

64.4

5.75

0.51

0.36

87.1


17


Page 18 of 39

Table 2: Textural and fluid transport properties of supercritically processed scaffolds. Fluid transport properties were obtained by 3D-modeling from the MIP-cumulative curves. Ds and kw denote the fractal dimension of the pore surface and the water permeability of the scaffolds, respectively.

Scaffold

PCL-0V-0Chit PCL-0V-10Chit PCL-1V-0Chit PCL-3V-0Chit PCL-5V-0Chit PCL-5V-5Chit PCL-5V-10Chit PCL-5V-15Chit

Pore size distribution, % vol. 100 µm µm µm 9.8 8.8 50.1 31.2 10.5 4.9 27.0 60.6 6.6 11.4 42.3 39.7 15.3 10.8 37.7 36.3 13.3 10.1 41.7 34.9 15.2 16.2 29.2 39.4 1.8 3.0 29.7 65.6 5.6 3.6 28.0 62.8

Interconnecti vity, %

Ds, -

kw·1012, m2

74.8 78.7 76.2 70.5 79.7 76.8 66.3 92.7

2.4 2.4 2.5 2.6 2.7 2.1

0.017 6.754 0.313 0.030 0.084 3.281 2.002 6.276

Cell infiltration, % 5 94 81 93 80 94 95 92

Pore size distributions obtained by MIP analysis highlight the pores with sizes of 10 µm and above as the major contribution to the overall pore volume for all the studied scaffolds (Table 2). The presence of vancomycin and mainly of chitosan in the scaffolds formulations resulted in a shift of the pore size distribution towards pore sizes above 100 µm. Namely, in the case of chitosan particles this shift was dependent on its content in the scaffold composition. The presence of particles in the scaffolds induced an heterogeneous nucleation of CO2 in the polymeric matrix that resulted in a higher macroporosity and was dependent on the particles content

33, 41

. Moreover, the observed trend of increasing pore roughness with higher chitosan

and/or vancomycin contents was confirmed by the increase in the fractal dimension of the pore surface (Ds, a value ranging from 2 to 3). A threshold for the Ds-value was observed with increasing chitosan content and was correlated to the previously mentioned segregation of the scaffolds components at 15 wt.% chitosan and above (Fig. 3d). The population of large macropores coupled to the high porosity observed for the PCL-based scaffolds are especially relevant to facilitate the cell attachment into the scaffold and the subsequent tissue growth

18

. However, along with overall porosity and macroporosity, pore

interconnection plays a key role in the promotion of tissue growth 42. The adequate combination of these properties should lead to an optimized porous 3D-structure that allows cell infiltration


18

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within the scaffolds as well as facilitates the transport of nutrients, metabolic products and wastes throughout the tissue. A 3D-simulation based on the mercury intrusion profiles from the scaffolds was carried out to predict the interconnection of these porous structures. These simulations were also employed to assess the scaffolds performance regarding both the transport properties of biological fluids and the capacity of human MSCs to percolate through the scaffolds. Pore interconnectivity was above 65 % for all tested scaffolds (Table 2). Scaffolds prepared with higher proportions in chitosan showed higher interconnectivity, reaching values up to ca. 93% for the PCL-5V-15Chit scaffolds. Nevertheless, the morphological interconnection of the porous structures is not only influenced by the pore interconnectivity extent but also by the pore throat size distribution, f.i. a scaffold with 100% interconnectivity will have poor biological fluid transport properties and cell infiltration capacity in case of presenting very narrow pore throat sizes or intricate pore-throat-pore paths. Accordingly, the permeability values of the scaffold formulations without chitosan had intricate 3D-structure resulting in poor water permeability values. Chitosan increased the water permeability more than two orders-ofmagnitude with respect to neat PCL scaffolds. The MSCs cell infiltration capacity was dramatically improved with the incorporation of both chitosan and/or vancomycin in the scaffolds turning from a virtually null capacity (PCL-0V-0Chit scaffolds) to a value close to full capacity (80-95 %). 3.2 Physicochemical characterization of PCL-based scaffolds PCL scaffolds showed a single melting peak. The Tm value of PCL increased after the supercritical foaming process if compared to PCLraw and was related to the rearrangement of crystallites upon the treatment 20. The incorporation of vancomycin in the scaffold formulations slightly reduced the melting point values and notably decreased the crystallinity of the PCL. This behaviour can be explained by a certain PCL-vancomycin interaction through hydrogen bonding between the carbonyl groups from PCL and the amino and hydroxyl groups from vancomycin

43

. The presence of chitosan

decreased the Tm value and increased the crystallinity of the PCL fraction. The role of chitosan


19


Page 20 of 39

as a secondary nucleation site for crystallization would promote the formation of crystals of smaller size and explains the observed changes in the thermal events

41

. The simultaneous

incorporation of vancomycin and chitosan resulted in a further decrease in the Tm values and intermediate PCL-crystallinity values in the 51-54% range. In this case, the observed thermal events can be explained by the chitosan-assisted secondary nucleation of PCL crystals counteracting the PCL-vancomycin chemical interactions.

Table 3: Thermal properties of PCL-based scaffolds processed under supercritical foaming conditions Material/scaffold

Tm (ºC)

∆Hm (J/g)

Crystallinity (%)

PCL-0V-0Chit

65.4±0.7

82.4±2.1

58.0±1.5

PCL-1V-0Chit

63.4±0.8

67.0±5.7

47.7±4.0

PCL-3V-0Chit

64.8±0.7

61.6±4.2

44.7±3.0

PCL-5V-0Chit

64.3±1.6

60.0±3.3

44.5±2.5

PCL-0V-10Chit

64.5±0.1

82.4±1.9

64.5±1.5

PCL-5V-5Chit

62.8±0.8

68.2±2.4

53.3±1.9

PCL-5V-10Chit

63.6±1.0

61.7±0.3

51.1±0.2

PCL-5V-15Chit

63.3±1.0

60.8±0.4

53.6±0.4

3.3 Vancomycin release Vancomycin-loaded PCL-based scaffolds showed two-stage release profiles: (i) a fast release during the first 8 hours followed by (ii) a slow and sustained release up to at least 2 weeks (Fig. 4,left). The modified vancomycin release in the scaffolds should be attributed to its incorporation to the scaffold matrix since vancomycin in the powdered form showed a complete dissolution in less than 2 hours in PBS medium (not shown). It should be noticed that vancomycin hydrochloride, which is highly water soluble (>100 mg/mL) was used in all experiments 44. The amount of vancomycin released from the scaffolds during the first release stage represented between 15 and 55 wt.% of the payload. Complete release of vancomycin was not attained in 2 weeks. The remaining vancomycin may be strongly interacting with the PCL matrix and would be released in a longer term simultaneously with PCL degradation, which


20

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takes place in the order of months

20

. The wellness of the fitting to a biexponential function

(Table 4) indicates that the mass transfer mechanism of vancomycin from the scaffolds was governed by the dissolution from two distinct fractions: a first fraction located on the outer surface of the large pores of the scaffolds and with faster dissolution, and the second fraction located in inner parts of the scaffolds and with a release from the PCL matrices governed by a complex interplaying diffusion and dissolution of the bioactive agent through the porous matrix45. The presence of chitosan increased the vancomycin release rate (Fig. 4,right) probably due to an enhanced wettability of the scaffolds facilitated by the increase in the pore interconnectivity and water permeability (cf. Section 3.1). Accordingly, the amount of vancomycin released during the first release stage (Dmax,1) from the chitosan-containing scaffolds was higher (Table 4). In the second stage, the amount of vancomycin released (Dmax,2) and its release rate (k2) increased with higher chitosan contents. The obtained vancomycin release profiles seem suitable to prevent bacterial colonization after implantation. The fast release stage might be effective in providing enough vancomycin to the surrounding environment to reach the required MIC values during the first 24-48 h after the surgical procedure, where there is a higher potential risk for bacterial colonization 4. Thereafter, the sustained release period during the following 2 weeks would be able to keep the vancomycin content above the MIC values during a mid-term period. The chitosan content in the scaffold formulation may be effective in tuning the vancomycin release rate to the specific demands.


21


Page 22 of 39

Fig. 4. Vancomycin release profile from PCL-based scaffolds in PBS pH 7.4 solution (37°C, 60 rpm) during 2 weeks: vancomycin (left) and chitosan (right) content effects. Legend: PCL-1V0Chit (white triangles), PCL-3V-0Chit (black triangles), PCL-5V-0Chit (white squares), PCL7V-0Chit (black squares); PCL-5V-5Chit (white diamonds), PCL-5V-10Chit (black diamonds) and PCL-5V-15Chit (white circles).

Table 4: Kinetic fitting parameters of the vancomycin release profiles from drug-loaded scaffolds in PBS solution (pH 7.4) according to Eq. (3). Dmax,1, dose%

k1, h-1

Dmax,2, dose%

k2·102, h-1

R2

PCL-1V-0Chit

15.6±0.5

0.906±0.092

14.6±1.0

0.798±0.158

0.995

PCL-3V-0Chit

46.1±2.2

1.191±0.182

39.1±3.0

1.180±0.301

0.987

PCL-5V-0Chit

34.8±1.3

1.176±0.149

26.4±2.9

0.773±0.234

0.987

PCL-7V-0Chit

40.5±2.0

0.966±0.158

65.0±14.6

0.379±0.152

0.988

PCL-5V-5Chit

54.5±1.3

1.311±0.108

43.3±2.5

0.877±0.148

0.995

PCL-5V-10Chit

42.9±2.0

0.953±0.133

52.3±2.8

1.062±0.186

0.994

PCL-5V-15Chit

48.8±1.4

1.292±0.122

44.4±1.9

1.229±0.175

0.996

Scaffold

3.4 Antimicrobial evaluation The scaffolds were designed to promote host cell attachment and to prevent bacterial colonization. Accordingly, the incorporation of an antibiotic in the formulation was expected to interfere with bacterial colonization avoiding biofilm formation and infectious perioperative episodes 46. The vancomycin-loaded scaffolds were evaluated regarding their ability to prevent biofilm formation (sessile population) and bacterial growth around the scaffolds (planktonic population) after 24 hours bacterial incubation. For S. aureus, vancomycin exhibited bactericidal performance on both sessile and planktonic populations regardless of the initial antimicrobial agent content in the scaffolds (1-7 wt.%). The attachment and proliferation of S. aureus was fully inhibited (Table 5) as also confirmed by SEM imaging (Fig. 5). Taking into account the vancomycin release rate from the scaffolds (Fig. 4) and the susceptibility of S. aureus to vancomycin, this experimental setup provided vancomycin concentrations in the medium after 24 h well above the minimum inhibitory


22

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concentration (MIC, 1-2 µg/mL 47) needed to kill the bacterial strain. The obtained antimicrobial effect of the scaffolds against S. aureus may be also extrapolated to other common Grampositive pathogens responsible for postoperative infections and with similar or lower vancomycin MIC values (e.g., S. epidermidis 48). Despite the intrinsic resistance of Gram-negative bacteria to vancomycin

11-12

, an antimicrobial

effect of the scaffolds against E. coli bacterial strain was obtained and was dependent on the initial vancomycin content in the scaffolds (Table 5 and Fig. 6). There was a reduction of sessile bacteria and a delay of planktonic bacterial growth compared with neat PCL-scaffolds. A slow growth of E. coli bacterial strain was observed but without loss of their viability, since the planktonic bacteria concentration after 24 h-incubation was higher than 0.1 of optical density (ca. 8 log10), close to the initial bacterial concentration used. The antimicrobial effect of chitosan was hindered due to the low solubility and the limited amount of positive charges of the polysaccharide at the pH (7.3±0.1) of the growth medium

49

, although a vancomycin-

chitosan synergistic effect on inhibiting E. coli bacterial adhesion and growth might also take place. Overall, the log reductions of 2 to 3 in sessile E.coli bacteria obtained for scaffolds containing more than 3 wt.% of vancomycin after 24 h of incubation is regarded as significant and of clinical relevance. Table 5: Quantification of planktonic and metabolically active sessile bacteria (S. aureus and E. coli) in contact with PCL-based scaffolds (37ºC, 24 h) by optical density and CFUs counting, respectively. Results were compared and equal letter (superscript) denotes statistically homogeneous groups (p

Antimicrobial properties and osteogenicity of vancomycin-loaded

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