(a) Through direction (d) (c)(b) Through direction Porous Three-Dimensional Scaffolds of Poly(3-Hydroxybutyric Acid) (PHB) and Poly(3-Hydroxybutyric-co-3-Hydroxyvaleric Acid) (PHBV) With An Improved Thickness As Cell Growth Supporting Materials Saiful Zubairi1, Alexander Bismarck1, Apostolis Koutinas2, Nicki Panoskaltsis3 and Athanasios Mantalaris1 1Department of Chemical Engineering, Imperial College London, 2Department of Food Science and Technology, Agricultural University of Athens, and 3Department of Haematology, Northwick Park & St. Mark’s campus, Imperial College London. For additional information please contact: email@example.com INTRODUCTION Objectives Over the past 30 years, polyhydroxy acids (PHA), particularly poly-3-hydroxybutyrate (PHB) and Rationale of this research copolymers of 3-hydroxybutyrate with 3-hydroxyvalerate (PHBV) have been demonstrated to be 1. To fabricate and optimize the suitable biomimetic scaffolds for culturing leukaemic cells ex vivo. suitable for tissue engineering applications. Specifically, these polymers have been used as a wound To promote the usage of POME in producing PHA via healing matrix and also as a wrap-around implant. However, to our knowledge, scaffolds from PHB 2. Study of CLL - lack of appropriate ex vivo models - mimic the ABNORMAL 3-D microbial fermentation process as an ADDED VALUE with thickness greater than 1 mm have not been produced yet. In this work, PHB and PHBV porous BM niches → To facilitate the study of CLL in its native 3-D niches. MATERIALS for Tissue Engineering applications. 3-D scaffolds with an improved thickness greater than 4 mm were fabricated and evaluated in terms of their physico-chemical characterization and cellular response on the Acute Myeloid Leukaemia cell line (HL-60) for 14 days. FIGURE 5: Efficacy of (A) salt-leaching process and (B) salt Efficacy of salt removal measured via ion conductivity and gravimetric analysis removal after lyophilization process via gravimetric analysis for NOVELTY * * PHB and PHBV (4%, w/v). *Significant difference with p<0.05 between the samples were highlighted by lines (n = 10). 120 Ability to fabricate porous 3-D scaffolds with an improved thickness greater than 4 mm from PHB and 110 99.67 99.97 PHBV without the presence of the etching surfaces and structural instability. 100 92.59 Effect of salt remnants in polymeric 3-D scaffolds on cell growth media 90 82.20 METHODOLOGY Solvent evaporation in fume cupboard (Complied with Effect of Porogen Residual On Cell Growth Media 80 FIGURE 6: Conductivity (κ) of cell growth media in the presence of scaffolds as a function of time at 20 ± 1 oC (n = 3). % Efficacy UK-SED, 2002: < 20 mg/m3) 70 Efficacy of Salt Removal 60 20.85 50 NS Polymer Concentration Vs. Thickness 20.8 40 Conductivity (mS/cm) Polymer solution in 30 20.75 organic solvent Polymer Concentration Vs. Time 20 10 20.7 Porogen-DIW leaching FABRICATION PHB (4%, w/v) porous 3-D scaffolds 0 20.65 Salt-leaching process Lyophilization process PHBV (4%, w/v) porous 3-D scaffolds (a) Polymer solution + Porogen 3 4 20.6 Control: Cell growth media without a scaffold Type of polyhydroxyalkanoates (PHAs) porous 3-D scaffolds 2 1 20.55 5 Porous 3-D PHB (4%, w/v) PHBV (4%, w/v) No lost of polymer mass Efficiency: PHB > PHBV → scaffolds throughout the SCPL process 20.5 Hydrophilicity: PHB > PHBV (b) 0 1 2 3 4 5 6 7 Polymer + Solvent Dried cast Polymer + + Porogen cast Porogen Time (days)(c)(a)(e) Conductivity of cell growth media = 20.77 mS/cm @ 20 ± 1 oC Porogen (i.e., NaCl, sucrose & etc.) Physico-chemical characterization FIGURE 7: Scanning electron micrograph FIGURE 1: of: (a) PHBV (4%, w/v) porous 3-D Schematic of the Solvent-Casting Particulate-Leaching (SCPL) process. The process comprises (1) mixing of polymer Through direction Through direction scaffolds vertical cross-section (35x); (b) (a) (c) solution with porogen; (2) adding the polymer solution with porogen into a Petri-dish and then incubated in the PHB (4%, w/v) porous 3-D scaffolds lyophilization flask to avoid development of etching surfaces; (3) evaporation of solvent for 48 h in the fume cupboard. The vertical cross-section 35x). The enlarged solvent evaporation is complied with the United Kingdom Solvent Emission Directive (SED), 2002 for Halogenated VOCs: views of (a) and (b) are shown in (c) and <20 mg/m3 (<≅ 12 kg of CHCl3); (4) leaching out porogen from dried cast polymer + porogen by using 10 liters of deionized (d) respectively (100x). Particles size: water for 48 h (changed twice/day) at 20 ± 1oC; (5) lyophilized porous 3-D scaffolds with the thickness greater than 4 mm; 212-850 µm. (6) A rectangular size of ∼10 mm x ∼10 mm x ∼5 mm porous 3-D scaffolds is incised prior to the physico-chemical characterization, in vitro degradation measurement and cellular proliferation studies. 16 FIGURE 8: Pore size distribution (PSD) PHBV (4%, w/v) porous 3-D scaffold 14 PHB (4%, w/v) porous 3-D scaffold of PHBV and PHB (4%, w/v) porous 3- Different concentrations of PHB and PHBV ranging from 1% to 5% (w/v) were prepared in chloroform. D scaffolds determined using mercury -dV/d(log D)/cm3/g Through direction Through direction 12 (d) Porous 3-D scaffold were fabricated using the Solvent-Casting Particulate-Leaching (SCPL) method. (b) intrusion porosimetry (MIP). 10 The efficacy of the SCPL method was determined using ion conductivity measurement and 8 gravimetric analysis (to determine any potential of polymer weight loss during the salt-leaching 6 process). The salt remnants left inside the scaffolds were measured using ion conductivity as an 4 ultimate validation prior to the physico-chemical characterization and cellular proliferation studies on 2 the Acute Myeloid Leukaemia cell line (HL-60). Analysis of statistical significance was performed using 0 one-way analysis of variance (ANOVA) test and Students t-test with a significance level of p<0.05. 0 50 100 150 200 250 300 350 400 Pore Diameter, D/µm RESULTS TABLE 1: Physical properties of PHB and PHBV Structural properties Polymeric porous 3-D scaffolds (4%, w/v) porous 3-D scaffolds. The table Polymer concentrations with respect to polymeric 3-D scaffolds thickness FIGURE 3: PHB (4%, w/v) PHBV (4%, w/v) summarizes the principal physical properties of two 5.5 Scaffold thickness, mm 5.25 ± 0.36 4.40 ± 0.52** polymeric porous 3-D scaffolds prior to the in vitro Kinetics of PHB and PHBV homogenization Ψ * * Pore size distribution Micro-pores 10 - 100µm + Micro-pores 10 - 100µm + cell proliferation studies. 5 process with respect to different (diameter: µm) Macro-pores 100 - 350µm Macro-pores 100 - 350µm concentration, % (w/v). (A): Inhomogeneous Polymers concentration, % (w/v) 4.5 Physical properties *Ψ (A) Inhomogeneous polymer solutions polymer solutions were occurred with the BET surface area, As, m2 g-1[a] 0.70 ± 0.02 0.82 ± 0.03* **(p<0.01) - Results are considered statistically significant (n = 10) as 4 contain glutinous semi-solid residual appearances of glutinous polymer materials compared with PHB. *(p<0.05) - Results are considered statistically 3.5 * Geometrical bulk density, g cm-3 0.084 ± 0.15 0.072 ± 0.28* significant (n = 4) as compared with PHB. [a] BET surface area (m2 g-1) = Ψ* at the bottom of the SCHOTT Duran Skeletal density, g cm-3[b][c] 0.47 ± 0.52 0.92 ± 0.14* Total skeletal surface area (m2)/skeletal mass (g). [b] ρs is the skeletal density * of the crushed scaffolds, which is determined from helium pycnometry. [c] 3 bottle. The mean values obtained from 10 Porosity, % 81.97 ± 1.22 92.17 ± 0.73* The higher pore volume (the higher the amount of absorbate intruded), the 2.5 experiments ± SEM are shown (n = 10). lower the skeletal volume. 2 * Ψ *Significant difference with p<0.05 for the * Poly(3-hydroxybutyric acid): PHB value changed as compared to the previous Solvent-cast thin film[a][b] TABLE 2: Wetting and wettability of water on 1.5 Surface physico-chemistry PHB PHBV Poly(3-hydroxybutyric acid-co-hydroxyvalerate): PHBV value. (Ψ) p<0.05 for solubility rate of PHB PHB and PHBV solvent-cast thin films surfaces. 1 Ψ vs. PHBV. (4%, w/v) (4%, w/v) 0.5 Contact angle, θapparent (deg.) 66.80 ± 0.2 79.24 ± 0.4* [a] Equilibrium contact angle on solvent-cast thin films on polypropylene sheet (n = 10). [b] Surface free energy, mN m-1 (γs) 54.13 ± 0.3 46.93 ± 0.2* Contact angle of polypropylene (PP) sheet without PHB and PHBV coating = 92.43 ± 0.3 o. [c] 0 Work of adhesive, mN m-1 (W SL)[c] +109.42 ± 0.2 +97.41 ± 0.3* (+) or (-) work of adhesive: A non-spontaneous or spontaneous process respectively. [d] (+) or (-) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 spreading coefficient: Water will spread or not spread over the surface respectively. *p<0.05 as Spreading coefficient (SH20/scaffolds)[d] -36.38 ± 0.3 -48.39 ± 0.2* compared with PHB. #p<0.05 as compared with apparent contact angle. Time of complete homogenization (mins ) TABLE 3: Melting temperature, heat of fusion at melting point FIGURE 2: Polymer[a][b][d] Melting Heat of fusion Crystalinity (%)[c] Replication (n = 10) and crystallinity of PHB and PHBV. Thickness of scaffolds at different polymer temperature at melting (∆Hm) concentrations. Measurement was done PHB 179.9 oC 104,974 J kg-1 71.9 using Digital Vernier Caliper (accuracy ± PHBV (12% PHV) 152.1 oC 72,708 J kg-1 49.8 [a] Thermal analysis data are provided by the Sigma-Aldrich. [b] Thermal analyses of PHB and Cut into 10 sections 0.01 mm). *Significant difference with p<0.05 PHBV are done using a model DSC-7 differential scanning calorimeter (Perkin Elmer, USA) under Porous 3-D a nitrogen atmosphere, at a heating rate of 10 oC min-1. [c] Crystallinity is determined using the as compared to PHB (n = 10). following heat of fusion values for 100 % crystalline materials: ∆H0, PHB =146,000 J kg-1. The ∆H0 scaffolds FIGURE 9: Kinetics of in vitro degradation process for PHB and PHBV (4%, w/v) for PHBV is assumed to be the same as that for PHB.[d] The degree of crystallinity, H* (%); of Randomly selected of 5 sections the polymer could thus be estimated by using the following equation: H* (%) = ∆Hm/∆H0 × 100 %. porous 3-D scaffolds are measured via mass analysis. The polymeric porous 3-D scaffolds are submerged in phosphate buffered saline (PBS) and incubated at 37 oC. Samples are periodically removed and dried under vacuum prior to analysis. (*) Average thickness p<0.05 for percent decreased from the previous value (n = 6). ΨSignificant difference Cell Proliferation Assay of Acute Myeloid Leukaemia Cell with p<0.01 between each polymers were highlighted by line (n = 6). Polymer General observation Thickness (mm) Line (HL-60) on Polymeric Porous 3-D Scaffolds concentration PHB PHBV 110 1% (w/v) Completely dissolved, homogenous solution appeared < 1.0 < 1.0 * % Residual weight of porous 3-D scaffolds 100 90 * * TABLE 4: Change of cell numbers on PHB and PHBV porous 3-D 2% (w/v) Completely dissolved, homogenous solution appeared < 1.0 < 1.0 scaffolds with time (by MTS assay). * * 80 * 3% (w/v) Completely dissolved, homogenous solution appeared 1.80 ± 0.79 1.60 ± 0.79* 70 4% (w/v) Completely dissolved, homogenous solution appeared 5.25 ± 0.36 4.40 ± 0.52* Ψ Incubation time Type of PHAs Cell number[a] 60 (days) (4%, w/v) 50 PHB (4%, w/v) porous 3-D scaffold (a) (b) (c) (d) 1 PHB 207,657 ± 76,869 PHBV (4%, w/v) porous 3-D scaffold 40 PHBV 136,182 ± 41,574 30 7 PHB 170,714 ± 105,416 20 PHBV 165,793 ± 133,283 PHB 5% (w/v) PHBV 5% (w/v) PHB 1% (w/v) PHB 3% (w/v) PHBV 3% (w/v) 10 14 PHB 195,000 ± 69,114 0 PHBV* 346,428 ± 32,732** 0 7 14 21 28 35 42 49 56 63 70 77 84 [a] Initial seeding 370,000 cells per sample. Time (days) **p<0.01 relative to day 1. *p<0.05 relative to PHB at day 14. FIGURE 4: Morphology of scaffolds at different polymer concentrations (a) Aerial view of PHB (5%, w/v), (b) Aerial view of PHBV (5%, w/v), (c) Aerial view of PHB (1%, w/v), (d) Aerial view of PHBV and PHB (3%, w/v). CONCLUSIONS (a) (c) FIGURE 5: Morphology of the polymeric porous 1. Polymer concentration of 4% (w/v) was considered an optimal concentration to produce an ideal porous 3-D scaffolds with a 3-D scaffolds in a rectangular shape with an thickness greater than 4 mm without the presence of etching surfaces and structural instability. approximate size of 10 × 10 × 5 mm3: (a) Aerial 2. High efficacies of salt-leaching process for both polymeric 3-D porous scaffolds were observed (99%, w/w) with no loss of view of PHB (4%, w/v), (b) Side view of PHB polymer weight throughout the process. PHB (4%, w/v) PHBV (4%, w/v) (4%, w/v), (c) Aerial view of PHBV (4%, w/v), (d) 3. The small amount of salt left inside the porous 3-D scaffolds might not give any adverse effect to the cell growth due to the Side view of PHBV (4%, w/v). electrolytes imbalance from the hypertonic media solution (excessive amount of salt in the cell growth media). 4. High in surface hydrophobicity → surface roughness + air trapped inside the pore grooves + contaminants of the salt on the pore surface. (b) (d) INNER SIDE 5. High in surface hydrophobicity → EXPECTED → low degree of cell attachment & proliferation (14 days of cellular response on INNER SIDE the AML cell line (HL-60)). PHB (4%, w/v) PHBV (4%, w/v) ∼10 mm ∼10 mm ACKNOWLEDGEMENTS INNER SIDE ∼ 5 mm The authors would like to thank the Malaysian Higher Education (MOHE), National University of Malaysia INNER SIDE (UKM) and the Richard Thomas Leukemia Fund for providing financial support to this project.