Improved multicellular response, biomimetic mineralization

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Biological and Medical Applications of Materials and Interfaces

Improved multicellular response, biomimetic mineralization, angiogenesis and reduced foreign body response of modified polydioxanone scaffolds for skeletal tissue regeneration Nowsheen Goonoo, Amir Wagih Fahmi, Ulrich Jonas, Fanny Gimié, Imade Ait Arsa, Sébastien Bénard, Holger Schönherr, and Archana Bhaw-Luximon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19929 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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ACS Applied Materials & Interfaces

Improved multicellular response, biomimetic mineralization, angiogenesis and reduced foreign body response of modified polydioxanone scaffolds for skeletal tissue regeneration Nowsheen Goonoo1,2*, Amir Fahmi3, Ulrich Jonas4, Fanny Gimié5, Imade Ait Arsa5, Sébastien Bénard6, Holger Schönherr1* and Archana Bhaw-Luximon2* 1Physical

Chemistry I, Department of Chemistry and Biology & Research Center of

Micro and Nanochemistry and Engineering (Cμ), University of Siegen, 57076 Siegen, Germany 2Biomaterials

Drug Delivery and Nanotechnology Unit, Centre for Biomedical and

Biomaterials Research, MSIRI Building, University of Mauritius, 80837 Réduit, Mauritius 3

Faculty Technology and Bionics, Rhine-Waal University of Applied Sciences,

Hochschule Rhein-Waal, Marie-Curie-Straße 1, 47533 Kleve, Germany

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

4Macromolecular

Chemistry, Department of Chemistry and Biology, University of

Siegen, 57076 Siegen, Germany 5 Animalerie, Plateforme

de recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte

Clotilde, Ile de La Réunion, France 6 RIPA,

Plateforme de recherche CYROI, 2 rue Maxime Rivière, 97490 Sainte Clotilde,

Ile de La Réunion, France *Corresponding

authors:

[email protected],

[email protected],

[email protected],

[email protected], [email protected] Abstract The potential of electrospun polydioxanone (PDX) mats as scaffolds for skeletal tissue regeneration was significantly enhanced through improvement of the cell-mediated biomimetic mineralization and multi-cellular response. This was achieved by blending PDX (i) with poly(hydroxybutyrate-co-valerate) (PHBV) in the presence of hydroxyapatite (HA) and (ii) with aloe vera (AV) extract containing a mixture of acemannan/glucomannan. In an exhaustive study, the behavior of the most relevant cell lines involved in the skeletal tissue healing cascade, i.e. fibroblasts, macrophages, endothelial cells and pre-osteoblasts, on the scaffolds was investigated. The scaffolds were shown to be non-toxic, to exhibit insignificant inflammatory responses in macrophages and to be degradable by macrophage-secreted enzymes. As a result of different 2 ACS Paragon Plus Environment

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phase separation in PDX/PHBV/HA and PDX/AV blend mats, cells interacted differentially. Presumably due to varying tension states of cell-matrix interactions, thinner microtubules and significantly more cell adhesion sites and filopodia were formed on PDX/AV compared to PDX/PHBV/HA. While PDX/PHBV/HA supported micrometer-sized spherical particles, nanosized rod-like HA was observed to nucleate and grow on PDX/AV fibers, allowing the mineralized PDX/AV scaffold to retain its porosity over a longer time for cellular infiltration. Finally, PDX/AV exhibited better in vivo biocompatibility compared to PDX/PHBV/HA, as indicated by the reduced fibrous capsule thickness and enhanced blood vessel formation. Overall, PDX/AV blend mats showed a significantly enhanced potential for skeletal tissue regeneration compared to the already promising PDX/PHBV/HA blends.

Keywords: Polydioxanone, poly(hydroxybutyrate-co-valerate), aloe vera, multicellular response, cell-mediated biomineralization, foreign body response 1.0 Introduction Skeletal tissue (bone, cartilage and tendon/ligament) defects represent a major burden on the worldwide healthcare system, in particular in view of the aging population. Tissue engineering scaffold-based reconstructive strategies offer exciting opportunities to overcome the reported poor self-healing capacity of skeletal tissue, which is a significant challenge in elderly patients1. However, the clinical translation of novel scaffold designs has not been very successful yet, mainly due to the absence of a simple, reproducible and effective approach, which is independent from the incorporation of cells, such as stem cells2. Skeletal tissue engineering (STE) aims at the use of temporary structures i.e. scaffolds to produce biological skeletal tissue substitutes as functional tissue replacement3. For instance, regenerated



cartilage can be derived by combining various cell types, such as chondrocytes and stem cells with scaffolds consisting of proteins, carbohydrates, synthetic materials, and composite polymers. On the other hand, repaired tendon/ligament using the principal elements of tissue engineering – cells, scaffolds, and bioactive molecules – has failed to restore the functional, structural, and biochemical properties of those of native tissue4. Skeletal tissue regeneration involves the family of connective-tissue cells, which are specialized for the secretion of collagenous extracellular matrix (ECM) and maintain the architectural framework of the body. Connective-tissue cells have a crucial role in support and repair of tissues and organs, as their differentiated character can respond to various types of damage5. Skeletal tissue regeneration in vivo occurs in several well-orchestrated steps and involves a range of different cell types, namely fibroblasts, endothelial cells, macrophages, osteoblasts, chondrocytes, and tenocytes6 (Scheme 1). A common initial inflammatory phase involves macrophages and other immune cells releasing cytokines followed by the recruitment, and proliferation of endothelial cells (ECs), which organize to form micro blood vessels (angiogenesis). These blood vessels are important for oxygen and nutrient diffusion as well as for the elimination of waste products. The next step for cartilage and bone repair involves the proliferation and interaction of fibroblasts and chondrocytes to form a fibrous cartilage. For bone restoration, this process is followed by the mineralization step, whereby osteoblasts mature and produce the inorganic mineral HA. For tendon regeneration, the repairing phase starts after the initial inflammatory phase, whereby tendon fibroblasts produce abundant collagen and ECM components, such as proteoglycans,, which are deposited at the wound site. The final stage for all skeletal tissue repair is the remodeling phase6.


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As reported in numerous previous studies, cells of different lineages require different microenvironments (topography, mechanical stiffness, hydrophilicity etc) for favorable adhesion7,8, proliferation9, migration10 and differentiation11,12,13,14, thereby increasing the complexity of finding adequate scaffolds for STE given that there are several cell types involved in the repair process. Scaffolds prepared by electrospinning are considered as attractive STE scaffolds, since the generated nanoscale three-dimensional (3D) fibers resemble the native ECM structures and provide a matrix with interconnected pores. The latter enhance molecular transport and surface area for better attachment osteogenic cells6,15. However, STE scaffolds are still faced by the challenge to simultaneously offer a conducive physical environment for cell attachment, sufficient mechanical strength, and trigger mineralization during the early stage of osteogenesis for functional bone formation. Electrospun scaffold-based STE suffers from a major limitation which is the inability to increase the loading of biologically active inorganic minerals due to a decrease in the overall solution viscosity16. Polydioxanone (PDX), best known for its clinical use as a monofilament suture17, was shown not to perturb the phagocytic functions of monocytes and neutrophils18. A simple way to improve mineralization of PDX scaffolds is via incorporation of a minor bioactive component within the scaffolds. The latter may be polysaccharides for instance kappa-carrageenan (KCG), fucoidan (FUC)19,20, chitosan21 or inorganic materials such as HA22; growth factors such as basic fibroblast growth factor (bFGF)23; adhesive Arg-Gly-Asp (RGD) peptide sequences24; peptides derived from osteogenic growth factors25 amongst others. Compared to pure PDX fibers, the incorporation of inorganic HA resulted in a more uniform and dense individual fiber mineralization when immersed in simulated body fluid (SBF) due to the presence of multiple



nucleation sites26. Additionally, PDX/HA was recently shown to induce better mineralization in vitro compared to poly(lactic-co-glycolic acid) (PLGA)/HA, whereby the acidic degradation products inhibited mineral growth26,16. PHBV, derived from microorganisms, has been extensively investigated for bone tissue engineering (BTE) applications due to its bioresorbability, and minimal inflammatory responses upon implantation27. The latter has been blended with other polymers in view of improving its low elastic moduli and poor osteoinductivity27. For instance, poly(L-lysine) surface modified electrospun PHBV mats displayed enhanced human fetal osteoblast cell adhesion, proliferation and differentiation as a result of higher wettability and surface roughness28. Recent studies probing into the bioactivity of AV indicated its high potential in tissue engineering (TE) applications by promoting fibroblast proliferation, migration, and enhanced collagen secretion29,30. AV gel contains over 75 different bioactive compounds including anthraquinones, polysaccharides, amino acids, enzymes, inorganic compounds etc31. In particular, the acetylated polysaccharide acemannan derived from AV gel was shown to be an effective bioactive agent for bone regeneration as confirmed by a significant increase in bone surface, bone volume and tissue mineral density in in vivo cavarial defects treated with this polymer32. Moreover, acemannan promoted soft tissue organization more specifically in vivo dentin formation by stimulating primary human dental pulp cell proliferation, differentiation, extracellular matrix formation, and mineralization33. In line with this study, the expression of osteogenic markers (alkaline phosphatase and osteocalcin), osteogenic differentiation as well as mineralization were enhanced in nanofibrous scaffolds consisting of polycaprolactone (PCL)/AV/silk fibroin compared to pure PCL34. In experimental wounds in rats, CarraSorb30, commercially available freeze-dried acemannan accelerated healing. This occurred through its macrophage-activating properties and


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ability to bind growth factors, thus stimulating granulation tissue formation. The same effect was found in open wounds and wounds with exposed bone in dogs through moist wound healing and autolytic debridement. CarraSorb30 is recommended to be used in the early inflammatory phase, particularly for moderately exuding wounds and wounds with exposed bone35.

Scheme 1: Orchestration and interplay of cells during the various stages of skeletal tissue healing



Thus, the successful development of clinically relevant scaffolds in STE requires in addition to an adequate choice of approaches, materials, processing parameters, the detailed knowledge how the different cell lines involved in the healing process are affected. As reported in this paper, scaffolds intended for STE applications were tested against a range of cell lines involved in the


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healing process in a comprehensive and systematic study for the first time. In addition to improving the multicellular responses as well as cell-mediated mineralization of PDX/HA via the incorporation of PHBV, the effects of PDX/AV electrospun blends on fibroblasts, macrophages, endothelial cells and pre-osteoblasts involved in the skeletal tissue healing cascade were compared to electrospun blends of similar PDX/PHBV ratio.

2.0 Experimental Section 2.1 Materials PDX (Resomer X 206S, inherent viscosity 2.0, Mw = 1.01  105 g/mol) was purchased from Evonik, Germany. PHBV (HV content 12 mol %) and nano-hydroxyapatite (HA) (approximate particle size = 100 nm and purity = 98.5%) were bought from SigmaAldrich and American Elements® (USA), respectively. 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) purchased from FluoroChem and Apollo Scientific was used as received. Milli-Q water and PBS solution used in this study was prepared as described in reference 19. AV powder was extracted from leaves of Aloe Barbadensis Miller grown at the Mauritius Sugar Industry Research Institute (MSIRI), Mauritius. Briefly, the AV gel was removed from the leaves and rinsed abundantly with water. The gel was soaked in distilled water at room temperature for one hour to remove the yellow exudate. The 9 ACS Paragon Plus Environment


colorless gel was then blended with a homogenizer and centrifuged at 3500 rpm for 30 minutes at 4°C. The supernatant was collected and precipitated in absolute ethanol (3× vol). The white precipitate obtained was collected by centrifugation at 3500 rpm for 15 minutes and was lyophilized for 48 hours at -40°C. L929 mouse fibroblasts (ECACC certified) were bought from Sigma Aldrich. The NIH3T3 fibroblast cell line was obtained from Dr. Jürgen Schnekenburger (Biomedical Technology Center of the Medical Faculty Münster, Germany). The Ea-hy926 endothelial cells and RAW blue 264.7 macrophages were purchased from ATCC. The SaOS-2 cells (human osteosarcoma cell line) were obtained from Dr. Ulrike Ritz (University Medical Center of the Johannes Gutenberg University Mainz, Germany). 2.2 Electrospinning PDX/PHBV was blended (100/0, 90/10, 80/20 and 70/30 w/w%) and HA was added at a concentration of 5 wt % (wrt to the total polymer mass) to all PDX/PHBV solutions. All polymer blend solutions were stirred for 24 hours before electrospinning. To prepare PDX/AV solutions, the polymers PDX and AV powder were dissolved together in HFIP and stirred at 300 rpm for 48 hours. 10 ACS Paragon Plus Environment

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Electrospinning was carried out at Rhine-Waal University of Applied Sciences, Kleve (Germany) and at CBBR (Mauritius) using an electrospinning machine purchased from IME Technologies (Netherlands) and Inovenso Company (Turkey) (bottom-up NE300 Laboratory scale electrospinner) respectively. The electrospinning parameters (Table 1) were optimized to produce continuous fibers, which were collected as non-woven fiber mats on a rotating grounded aluminum target (1500 rpm). The electrospun scaffolds were stored in a desiccation chamber until further analysis. Table 1: Electrospinning parameters used for PDX/PHBV/HA and PDX/AV solutions

Blend system

Solvent

Concentration

Flow rate /

Voltage

Air-gap

system

/

(mL/hour)

/

distance /

(kV)

(cm)

(mg/mL) PDX/PHBV/HA

HFIP

100

3.5

+26

15

PDX/AV

HFIP

100

0.3

+20

15

2.3 Characterization of electrospun mats The average fiber diameter of the electrospun mats was determined by SEM as reported previously19. The pore area of the electrospun mats were measured by 11 ACS Paragon Plus Environment


analyzing SEM images via ImageJ software. Briefly, the images were thresholded and the plugin “Analyze Particles” was used. 50 pore area measurements were noted for all electrospun blend mats. The SEM images were taken using a CamScan microscope (CS24, USA) (University of Siegen, Germany) and a Tescan Vega 3 LMU microscope (CBBR, Mauritius) with accelerating voltages of 25 and 30 kV`, respectively. High resolution field emission (FE)-SEM images were acquired using a Quanta 450 ﬁeldemission-scanning electron microscope equipped with a Thermo Scientific system of energy dispersion X-ray microanalysis (EDX) (University of Siegen, Germany). Samples were prepared as reported in reference 20. The thermal properties of the electrospun blend fibers were analyzed by differential scanning calorimetry (DSC) and a thermogravimetric analyzer (TGA), as reported previously19. The contact angles of the electrospun mats were measured using Milli-Q water as a probe liquid with a Krüss Drop Shape Analyzer DSA 25 (Advanced Lab GmbH, Germany). Static contact angles were measured as reported in reference 20.


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1H-

NMR analysis was performed on a 600 MHz Avance III Bruker NMR spectrometer

equipped with a

1

H/

19

F,

13C

and

15N

cryoprobe. 1H NMR spectrum was recorded at

room temperature using in D2O (Eurisotop, France) as solvent using a sweep width of 20 ppm and 512 scans. 2.4 Culture of NIH3T3 mouse fibroblast cells, L929 mouse fibroblasts, murine RAW 264.7 macrophage cells, Ea-hy 926 endothelial cells and SaOS-2 pre-osteoblast cells The NIH3T3 and SaOS-2 cells were cultured at standard conditions (37°C, 5% CO2) as reported earlier20. L929 mouse fibroblast cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM Lglutamine and 1% penicillin/streptomycin at 37°C and 5% CO2. The culture medium of RAW 264.7 macrophages consisted of RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin, 0.5 g/mL amphotericin B, 1 mM sodium pyruvate and 2 mM L-glutamine. The human endothelial Ea-hy926 cells Modified

Eagle’s

Medium

(DMEM)

were cultured in Dulbecco’s

supplemented

with

10%

FBS,

1%

penicillin/streptomycin and HAT (hypoxanthine 100 µmol/L; aminopterin 0.4 µmol/L and thymidine 16 µmol/L) at 37°C in a 5% CO2 humidified atmosphere. 13 ACS Paragon Plus Environment


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2.4.1 Live-dead staining Live-dead staining was performed as previously reported in reference 19. The number of live and dead cells was then counted and the cell viability was calculated according to Equation 1. 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠

Cell viability (%) = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 + 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 × 100

Equation 1

2.4.2 Dehydration of scaffolds for SEM analysis Cell-seeded scaffolds as well as scaffolds removed from rats after 2 weeks were fixed for SEM analysis as reported in references 19, 20 . 2.4.3 MTT Assay The MTT assay was conducted as reported earlier on days 3 and 7, respectively20. 2.4.4 Investigation of the innate inflammatory response induced by the scaffolds onto RAW 264.7 macrophages and macrophage mediated degradation Electrospun mats were disinfected with ethanol as reported before and the mats were seeded with 25,000 RAW 264.7 cells/well (96 well-plate).


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The RAW 264.7 cells seeded onto the scaffolds were stained using Hoechst (for the nucleus) and Phalloidin-Alexa 488 (for actin) on Day 3. Briefly, the cell seeded scaffolds were washed with PBS, fixed with paraformaldehyde (4% in PBS, 30 minutes; VWR), permeabilized with Triton X-100 (0.2% in PBS, 10 minutes; VWR), and washed with PBS (3 times) at room temperature. Samples were incubated in a solution of Alexa Fluor™488 Phalloidin for 30 minutes (300 U/mL in 1% BSA; Invitrogen, Life Technologies) as well as DAPI staining solution (Sigma-Aldrich) for 15 minutes. Samples were mounted with Ibidi mounting medium (Germany) on cover glass slides and attached to microscope slides. Furthermore, the RAW 264.7 seeded scaffolds were fixed for SEM analysis and SEM images were taken as reported in Goonoo et al20. The extent of F-actin-rich membrane protrusions (ruffling index) was scored using a scale of 0-3 [modified from reference 36], where 0 = no protrusion, 1 = protrusions in one area of the cell, 2 = protrusions in two distinct areas of the cell, 3 = protrusions in more than two distinct areas of the cell. The ruffling index was calculated as the average of protrusion scores of at least 50 cells36).



Macrophage mediated degradation of electrospun scaffolds was investigated by culturing RAW 264.7 cells on the scaffolds. After 2 weeks, the scaffolds were washed with PBS (3×) and placed in 10% Triton X-100 for 48 hours. The latter were then washed several times with distilled water to remove all traces of Triton and finally air dried and sputter coated for SEM.

2.4.5 Cell migration assay Cell migration assay was performed as reported previously in reference 37. After 24 h incubation, the scaffolds were removed from the well plate and the cells were stained using FDA and visualized using a fluorescence microscope (Evos® FL Imaging System). The number of migrating cells was quantified via ImageJ. 2.4.6 Investigation of the mineralization of SaOS-2 cells by SEM and Alizarin Red-S staining Mineralization of the cells was induced and assessed as reported earlier20. Furthermore, SEM images of the mineralized scaffolds were taken.


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2.5 In vivo biocompatibility studies 2.5.1 Implantation surgery Electrospun fiber mats were subcutaneously implanted into 9-week-old, male and female Wistar rats (Janvier Labs, Saint Berthevin, France) for 2 weeks. Five Wistar rats were used for the study. Animal surgeries and care were performed following guidelines approved

by

the

Ethical

Committee

of

Réunion

Island,

protocol

APAFIS#16368_2018052311219125_v3. Food and water was provided to the rats ad libitum. Electrospun scaffolds (1.0×0.5 cm2) were soaked in 70% ethanol overnight. Ethanol was allowed to evaporate on the next day in a sterile cell culture hood and the dry scaffolds were rinsed three times with PBS for 5 mins each and finally few drops of normal saline was added to the scaffolds prior to implantation. The rats were anesthetized using isoflurane (Isoflo® 100%, Zoetis,) throughout the surgery. The dorsal region of the rats was shaved, and disinfected with 70% ethanol followed by betadine solution. The disinfection procedure was repeated 3 times. Buprenorphine (0.05 mg/kg) was then injected subcutaneously. Four slits (each 1 cm 17 ACS Paragon Plus Environment


long) at equal distances (2 cm) from the spine were cut through the skin using a sterile scalpel blade so that subcutaneous pockets were created. Three layers of each scaffolds (average overall thickness= 1 μm) were then inserted into the pockets using a pair of micro-dissecting forceps. The skin was sutured with intradermal suture (PGLA 4/0 suture) to close the pockets. The area was disinfected with betadine solution and the rats were allowed to recover from anesthesia. Males and females were housed in separate cages. Post-op verification was made every day to check for evidence of wound complications, such as redness, infection, seroma, abscess, hematoma, or skin dehiscence. A graphical illustration of the surgical procedure is provided (Figure S8 A). After 2 weeks, the scaffolds were removed and fixed for SEM analysis. Furthermore, tissue sections were removed from all the rats and stained using Masson’s Trichrome. The scaffolds are labelled as follows: upper layer, middle layer and lower layer. Afterwards, the rats were euthanized with an intraperitoneal injection of pentobarbital (100 mg/kg). 2.5.2 Histology and image analysis Following euthanasia, the implant sites with surrounding tissue were removed and immersed in 4% PFA solution for 24 h, followed by immersion in 70% ethanol overnight.


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Sections were taken for routine paraffin embedding (sectioned at 5 μm) and the histology slides were stained with Masson’s Trichrome. Histological stained slides were examined using light microscopy (Nanozoomer 560 digital slide scanner, Hamamatsu). Thickness of fibrous capsules was measured from Masson’s Trichrome stained images. The number of vascular structures at a distance of 100 μm from the scaffold surface was counted. Only blood vessels with intact lumen were taken into account. 2.6 Statistical Analysis The data are presented as arithmetic mean ± standard error of mean. Statistical analyses were done with the two-way analysis of variance (ANOVA) test (Graph Pad Software, San Diego, CA, USA) with the blend system and composition as the two varying factors. A one-way analysis of variance (ANOVA) test (OriginPro 8, Origin lab Corporation, USA) was used for statistical analysis of data obtained from in vivo studies, with the blend system as the varying factor. A value of p < 0.05 was considered statistically significant.

3.0 Results and Discussion 19 ACS Paragon Plus Environment


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The scaffolds and their properties were systematically studied and their interaction with the cell lines important for the skeletal tissue healing cascade unraveled. This section presents (i) the fabrication, characterization and comparison of the physico-chemical properties of blends of PDX/PHBV/HA v/s PDX/AV (ii) in vitro multicellular responses of PDX/PHBV/HA blends and (iii) in vitro and in vivo bio-performance comparison of electrospun mats of PDX/PHBV/HA v/s PDX/AV.

3.1

Fabrication,

physico-chemical

characterization

and

comparison

of

fibrous

PDX/PHBV/HA vs PDX/AV mats PDX/HA was blended with PHBV with the weight ratio of PHBV to PDX varying from 0 to 30 w/w % while the concentration of HA was kept constant at 5 wt% with respect to the total mass of the polymers. It is expected that such a blending would lead to less hydrophilic materials, which favor cell proliferation compared to very hydrophilic ones38,39. A PHBV content beyond 30 w/w% in the blend brought about a drastic decrease in solution viscosity, which impeded electrospinning. SEM images of the resulting electrospun blend fibers showed bead-free morphology (Figure S1) with fiber diameters ranging between 0.63 – 0.98 µm (Table 2). The variation among the fiber diameters and pore areas was small for the various PDX/PHBV/HA blends. Table 2: Summary of fiber diameter and pore area

Blend

Fiber diameter/


Pore area/

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composition

µm

µm2

100/0/5

0.98 ± 0.3

1.84 ± 0.9

90/10/5

0.91 ± 0.3

2.37 ± 0.9

80/20/5

0.63 ± 0.3

2.11 ± 0.9

70/30/5

0.78 ± 0.3

2.11 ± 0.6

PDX/PHBV/HA

The practically unchanged melting temperature of PDX and PHBV phases of electrospun PDX/PHBV/HA blend fibers indicated polymer immiscibility (Table S1). However, PDX acts as an anti-nucleating agent in the blend system impairing the crystallization of PHBV as may be deduced from the increase in the degree of crystallinity of PHBV with decreasing PDX content in the blends. Furthermore, data obtained from thermogravimetric analysis supported immiscibility of the PDX/PHBV/HA system, as evidenced by the presence of two distinct degradation stages with the onset degradation temperatures of the two stages being very close to that of PDX/HA and PHBV (Table S2). To further investigate the effect of blend ratio on the wettability of the blend mats and hence, the surface distribution of the polymers within the blend fibers, the water contact angles of the electrospun mats were measured. The data from Table 3 suggest that increasing addition of



PHBV to electrospun PDX/PHBV/HA mats led to enhanced presence of hydrophobic PHBV at the surface of the blend fibers.

Table 3: Water contact angle data of electrospun PDX/PHBV/HA mats Samples (w/w/w %) PDX PHBV PDX/PHBV/HA 100/0/5 PDX/PHBV/HA 90/10/5 PDX/PHBV/HA 80/20/5 PDX/PHBV/HA 70/30/5

Contact angle/ ° unable to measure reliablya 112.8 ± 1.0 41.2 ± 2.7 107.1 ± 1.9 121.3 ± 1.3 129 ± 3.2

a The water contact angle of pure PDX mat could not be measured reliably since the water droplet was absorbed too quickly

(within first 5 seconds).

AV gel was extracted from the leaves and lyophilized as detailed in the experimental section. The 1H-NMR spectrum of the AV gel powder extracted from AV leaves indicated that the latter consisted mainly of glucose (α-glucose at 4.6 ppm and β-glucose at 5.2 ppm), malic acid (2.5, 2.7 and 4.45 ppm), acemannan (2.0-2.3 ppm) and other polysaccharides (3.4- 4.5 ppm) (Figure S2)40 and showed the absence of lactic acid (1.34, 4.27, 11 ppm) and succinic acid (2.5 and 11 ppm), which are indicators of bacterial fermentation and enzymatic degradation, even after prolonged storage at low temperature. A 70/30 PDX/AV blend was prepared in line with the maximum PHBV content achieved in PDX/PHBV/HA mats for comparison. The 70/30 PDX/AV electrospun blend mat resulted in average fiber diameter and pore area of 0.28 ± 0.1 and 0.61 ± 0.35 µm2 respectively. The main aim was to compare the effects of PHBV/HA vs AV in improving the multicellular responses involved in the skeletal tissue healing process.


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Thermal analysis of the PDX/AV blend system indicated partial miscibility of PDX and AV compared to complete immiscibility of the PDX/PHBV/HA system (Tables S1 and S2). Indeed, the onset degradation temperature of PDX/AV shifted to higher temperatures compared to PDX/PHBV/HA. From DSC results, it could be deduced that AV acted as a nucleating agent to PDX, favoring the formation of crystalline domains, as suggested by significantly higher values of crystallinity of PDX/AV compared to pure PDX. Furthermore, the 70/30 PDX/AV blend mat showed improved wetting behaviors (46.4° ± 2.1) compared to 70/30 PDX/PHBV/HA (129° ± 3.2).



3.2 Orchestration of skeletal tissue repair - Fibroblasts, macrophages, endothelial cells and pre-osteoblasts responses on electrospun PDX/PHBV/HA blend scaffolds As highlighted in the introduction, several cells are involved in the skeletal tissue repair process and hence to investigate the skeletal tissue regeneration potential of the fabricated materials, it is paramount to evaluate the cell-biomaterial interaction of several cell lines. In the sub-sections, which follow, we discuss in details the multi-cellular responses of PDX/PHBV/HA blend mats for STE. In particular, the effects of the materials on fibroblasts, macrophages, endothelial cells and pre-osteoblasts will be discussed (Scheme 2). Scheme 2: Investigated scaffold-cell interaction


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3.2.1 Preliminary biocompatibility of electrospun mats: Cell viability and proliferation of NIH3T3 fibroblasts A crucial requirement for TE scaffolds is that the latter must be non-toxic and should support cell attachment and proliferation. As confirmed from live-dead staining of NIH3T3 cells on PDX/PHBV/HA mats, the incorporation of PHBV within PDX/HA scaffolds did not significantly reduce the cell viability with all blend scaffolds showing high cell viability (Figure 1A). To further investigate the influence of varying blend ratio on cell proliferation, NIH3T3 cells were cultured on PDX/PHBV/HA scaffolds for a period of 3 (Figure S3) and 7 (Figure 1B) days and the cell proliferation was assessed using the MTT assay. Clearly, the incorporation of PHBV within PDX/HA fibers significantly favored NIH3T3 cell proliferation, as was noted from the drastic increase in relative absorbance values (Figure 1B). Overall, it can be deduced that addition of PHBV within PDX/HA scaffolds led to enhanced fibroblast proliferation with a more prominent enhancement in the 70/30/5 PDX/PHBV/HA scaffold. These results prompted testing of other cell lines on these scaffolds.



Figure 1: (A) Relative cell viability of NIH3T3 on PDX/PHBV/HA and PDX/AV scaffolds after 24 hours; (B) MTT assay results following NIH3T3 culture on PDX/PHBV/HA and PDX/AV fibers on day 7. All values from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p < 0.0001 and (ns) not significant.

3.2.2 In vitro innate inflammatory response induced by the scaffolds and macrophage mediated degradation of scaffolds The morphology of macrophages cultured on the electrospun mats was investigated to assess the host response for subsequent in vivo implantation. Following seeding onto electrospun scaffolds, the cells were found to maintain a rounded morphology on all scaffolds. This observation is indicative of insignificant cellular activation, as seen in SEM (Figure 2A-B) and fluorescence micrographs (Figure S4). 26 ACS Paragon Plus Environment

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However, with the addition of PHBV to PDX/HA, the presence of significant cell surface protrusions probing for the presence of phagocytic targets i.e. ruffles were noted (Figure 2). Similar macrophage responses have been reported due to addition of lipopolysaccharides41. The sequential activation of signaling pathways coordinating actin-driven formation of plasma membrane protrusions (ruffles) and circular ruffles (macropinocytic cups)42

results in the

formation of these protrusions. Ruffling indices of cells on each electrospun mats were calculated by determining the average of the ruffling scores (minimum 0 and maximum 3) of at least 40 cells. Results indicated that addition of PHBV enhanced the cell’s ability to undergo Factin-enriched membrane protrusions in RAW 264.7 cells (Figure 2E). A higher magnification of the ruffling region on the cell surface shows the openings of macropinocytic cups or pockets (Figure 2D).



Figure 2: SEM images of RAW 264.7 cell morphology on PDX/PHBV/HA (A) 100/0/5 (B) 70/30/5 and (C) PDX/AV 70/30, (D) higher magnification SEM image of macrophage and (E) summary of ruffling indices of RAW 264.7 cells seeded on electrospun PDX/PHBV/HA and PDX/AV mats. All values from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p < 0.0001 and (ns) not significant.

Macrophage mediated degradation of the scaffolds was studied to address the need for STE scaffold to degrade in vivo. In this respect, the macrophage mediated degradation of the scaffolds was studied following 2 weeks post culture with RAW 264.7 cells. After 2 weeks in culture with the cells, all scaffolds started losing their fibrous integrity and showed signs of extensive degradation via fiber melting (Figure 3).


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Figure 3: SEM images depicting scaffold architecture of (A) 100/0/5 PDX/PHBV/HA (B) 70/30/5 PDX/PHBV/HA (C) 70/30 PDX/AV after 2 days (D) 100/0/5 PDX/PHBV/HA (E) 70/30/5 PDX/PHBV/HA (F) 70/30 PDX/AV after 2 weeks

3.2.3 Morphology and spreading of endothelial Ea-hy926 cells To assess whether the PDX/PHBV/HA mats can act as a good substrate for endothelial cell (ECs) adhesion and support subsequent physiologic processes, such as vascular development and angiogenesis, ECs were seeded onto PDX/PHBV/HA for up to 7 days. The Ea-hy 926 cells were



shown to adopt an elongated morphology, which is typical of healthy endothelial cells (Figure 4). This indicates that the scaffolds did not induce significant illicit responses to the cells. In addition, the cells displayed projections, i.e. sprouts, connecting with similar projections from other cells forming cell-cell contacts on all scaffolds except on 100/0/5 and 90/10/5. Some cells also rearranged to form capillary-like tube structures, as is noted from Figure 4F and Figure S5. The formation of these structures has been correlated with angiogenic potential of the underlying substrate43. Based on these preliminary results, PDX/PHBV/HA 80/20/5 and 70/30/5 appear to be promising materials that can induce angiogenesis.

Figure 4: Fluorescence microscopy images of Ea-hy926 cells on PDX/PHBV/HA (A) 100/0/5 (B) 90/10/5 (C) 80/20/5, (D) 70/30/5 (E) PDX/AV 70/30 and (F) SEM image of PDX/PHBV/HA 70/30/5 showing capillary-like tube structure after 7 days


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3.2.4 Cell-electrospun blend mat interactions: Mechano-sensing of mouse fibroblasts Mouse fibroblasts cultured on the electrospun scaffolds adopted different morphologies as noted from the SEM images (Figures 5A-C). In particular, the cells proliferated as spindle-shaped on electrospun PDX/PHBV/HA 100/0/5 scaffolds while they adopted a flat elongated shape on the 70/30/5 scaffold. This may be rationalized by differences in the physico-chemical properties of the scaffold materials44. Increasing amounts of actin filaments were noted with increasing PHBV content on PDX/PHBV/HA mats (Figure 5D). Microtubules connecting cell to cell were observed on all electrospun mats. Interestingly, the diameter of the microtubules increased from 0.37 to 0.68 µm with increasing PHBV content (Figure 5J).



Figure 5: SEM images of fibroblasts on PDX/PHBV/HA (A) 100/0/5 (B) 70/30/5 (C) PDX/AV 70/30 (D) extensive formation of actin filaments (E) high magnification image 32 ACS Paragon Plus Environment

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depicting EVs and filopodia (FLP) on the surface of fibroblasts, (F) presence of EVs around fibers (G) formation of circular “holes” in between cells (H) presence of EVs within circular “holes” (I) degradation of scaffold via fiber melting and (J) summary of microtubule diameter on electrospun scaffolds. All values from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p < 0.0001 and (ns) not significant.

3.2.5 Migration of L929 fibroblast cells – Damage repair simulation The ability of PDX/PHBV/HA mats to act as a support to facilitate cell migration was examined by performing the scratch assay. As shown in fluorescence microscopy images (Figure S6), the gaps in the monolayer covered by PDX/PHBV/HA 80/20/5 were significantly narrower than the remaining scaffolds revealing significant closure of the scraped area. Furthermore, quantification of the number of migrated fibroblasts within the scratched area revealed significantly higher number of cells for the 80/20/5 PDX/PHBV/HA mats (Figure 6). These observations indicate that the scaffolds can provide support to repair damaged tissue via cell-cell and cell-matrix interactions.



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Figure 6: Number of migrated fibroblast cells on PDX/PHBV/HA and PDX/AV mats. All values from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p < 0.0001 and (ns) not significant.

3.2.6

Differentiation

of

pre-osteoblasts

and

cell-mediated

biomineralization

of

hydroxyapatite Cell morphology Following differentiation of pre-osteoblast cells (SaOS-2) into osteoblasts on the electrospun scaffolds, discernible differences in cell morphology could be noted between PDX/PHBV/HA 100/0/5 and the other blend compositions (Figure 7). Indeed, SaOS-2 cells grown on 100/0/5 mat showed limited spreading (which is typical of the first phases of substrate adhesion) in contrast to cells on the other blend mats, which displayed polygonal shapes and extended filopodia. The


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length of filopodia of SaOS-2 cells varied, as summarized in Figure 7F, with the 80/20/5 PDX/PHBV/HA composition, resulting in the formation of longer filopodia. Together, these results indicate that cells can sense through specific surface receptors for adhesive ligands, the chemical structure and the micro and nano-scale morphological properties. This resulted in more cell adhesion sites (filopodia) in the blend mats containing PHBV and lowest cell spreading on PDX/PHBV/HA 100/0/5 mat. This suggests that the blend scaffolds act as a promising substrate material favoring pre-osteoblast attachment and differentiation.



Figure 7: SEM images depicting SaOS-2 cell morphology on PDX/PHBV/HA (A) 100/0/5 (B) 90/10/5 (C) 80/20/5 (D) 70/30/5 and (E) PDX/AV 7030 after 7 days culture in osteogenic differentiation medium (F) summary of the length of filopodia of SaOS-2 cells v/s blend composition. All values from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p < 0.0001 and (ns) not significant. 36 ACS Paragon Plus Environment

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SaOS-2 cell-mediated biomineralization of hydroxyapatite SaOS-2 cells readily formed mineralized nodules, when cultured on all PDX/PHBV/HA mats. Mineralized nodules were noted on the surface of SaOS-2 cells as well as on fibers (Figure S7), suggesting that the blend fibers stimulated extensive SaOS-2 cell-mediated mineralization. Elemental analyses using energy dispersive X-ray (EDX) analysis showed that the Ca/P ratio in the mineralized nodules on PDX/PHBV/HA fibers was close (1.61) to that of HA (1.67) (Figure 8).

It has been proposed that mineralization by cells is initiated by matrix vesicles. The latter are cell-derived ECM enclosed particles, of about 0.1–1 μm in diameter. Matrix vesicles have been shown to possess a unique composition, distinct from the plasma membrane, which facilitates the initiation of matrix mineralization45. They can also increase phosphate concentration due to high TNAP activity46. Inorganic phosphate can be subsequently channeled into vesicles by a sodiumdependent phosphate transporter47. Matrix vesicles are highly enriched in annexin V and phosphatidylserine48, facilitating the influx of calcium into the vesicles. The high calcium concentration, inorganic phosphate and phosphatidylserine induces insoluble CaPO4phosphatidylserine complex formation at the inner leaflet of the matrix vesicle membrane48. The presence of matrix vesicles was noted on almost all SaOS-2 cell surfaces (Figures 9A and B). Interestingly, the matrix vesicles contained less calcium (Ca/P=0.15) compared to the nodules formed on the fibers (Ca/P=1.61) (Figure 8).



The amount of mineralized calcium nodules formed was quantified indirectly using the Alizarin Red-S staining (Figures 9D & E). No significant increase in absorbance values was noted among the blends of PDX/PHBV/HA after both 7 and 14 days.


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Figure 8: EDX analysis of mineralized nodules on (A) PDX/PHBV/HA 70/30/5, (B) PDX/AV 70/30 and (C) matrix vesicles formed by SaOS-2 cells



Figure 9: SEM image of matrix vesicles formed on SaOS-2 cell surfaces at (A) low magnification (B) high magnification, (C) summary of nodule diameter of particles formed during the cell-mediated biomineralization process, (D) Absorbance following Alizarin-Red S staining (for hydroxyapatite deposition by SaOS-2 cells) after 7 days and (E) after 14 days. All values from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p < 0.0001 and (ns) not significant.

3.3 Bio-performance comparison of the effects of adding PHBV/HA vs AV in electrospun PDX (A) In vitro multicellular responses: Fibroblasts, macrophages, endothelial cells and preosteoblasts Due to significant differences in miscibility and wetting of PDX/PHBV/HA and PDX/AV, it was hypothesized that cellular responses of the 2 blend systems will be different. The 70/30 PDX/AV blend fibers resulted in practically similar relative cell viability values as the 70/30 PDX/PHBV/HA mats with only a slight increase for the PDX/AV system (Figure 1A). However, MTT assay following culture of fibroblasts on the two scaffold materials for 7 days showed that the PDX/PHBV/HA mat led to relative absorbance values, which were almost three times higher than that of PDX/AV mat (Figure 1B). Similarly to the 70/30 PDX/PHBV/HA, the 70/30 PDX/AV mat did not induce significant activation of RAW 264.7 mouse macrophage cells, as shown by the round cell morphology (Figure 2 and S3). Moreover, both blend scaffold mats resulted in ruffling of macrophages cells with the PDX/AV mat, leading to a slightly higher ruffling index (Figure 2E). Compared to PDX/AV, the PDX/PHBV/HA mat displayed higher stability towards degradation by 40 ACS Paragon Plus Environment

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macrophage secreted enzymes (Figure 3). Ea-hy926 endothelial cells adopted healthy elongated shapes on both PDX/PHBV/HA and PDX/AV mats with the formation of tube-like structures (Figure 4). This observation suggests that both blend mats are promising materials for inducing angiogenesis. Mouse fibroblast cells adopted both elongated and dendritic shapes on the PDX/AV mat while on PDX/PHBV/HA they were mostly polygonal shaped. Compared to the fibroblasts on PDX/PHBV/HA, the ones on PDX/AV seemed to be in a state of high tension as indicated by the presence of an extensive actin filament network (Figure 5D), filopodia (FLP) and extracellular vesicles (EVs) (Figure 5E) emerging from the fibroblast surface through an active process of budding from the filopodial tips. EVs shed from the cell surfaces were also found within the fibrous scaffold surrounded by a network of fibers (Figure 5F). The fibroblast cells were found to undergo a rearrangement so as to form circular “holes” with the fibrous scaffold in the middle (Figure 5G). Interestingly, this phenomenon was noted only in PDX/AV scaffold. The cells around these “holes” produced EVs, which then migrated into the holes (Figure 5H). In addition to ligand–receptor interaction, surface recognition of specific cell adhesion molecules, or transfer of cytoplasmic components through junctional coupling, cells also interact through the release of EVs49. These vesicles may act as carriers of MMP-9, which are then released into the cell culture medium to degrade the matrix50. In this study, SEM images confirmed PDX/PHBV/HA and PDX/AV scaffold degradation via fiber melting (Figure 5I) suggesting that the vesicles may contain enzymes to degrade the scaffold paving the way for cell infiltration. However, significantly more extensive network of actin was noted on PDX/AV scaffolds.



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In contrast to 70/30 PDX/PHBV/HA scaffold mats, thinner microtubules were formed on the 70/30 PDX/AV mat (Figure 5G). It is known that the function of microtubules generated by fibroblasts vary depending on the tension state of cell-matrix interactions51. At a low tension state i.e. absence of stress fibers, microtubules assist in cell spreading while at a high tension state, microtubules are required for cell polarization but not for spreading. It is hypothesized that thicker microtubules facilitating cell spreading were noted for 70/30 PDX/PHBV/HA mats requiring mechanical strength compared to thinner ones on 70/30 PDX/AV, which were probably for cell polarization only. In vitro scratch assay performed to simulate damage repair showed the higher potential of PDX/AV compared to the corresponding 70/30 PDXPHBV/HA. Indeed, higher number of cells migrated into the scratch (wounded area) for the PDX/AV (Figure 6). Pre-osteoblast cells on PDX/AV showed high cell spreading with higher number of adhesive structures compared to PDX/PHBV/HA (Figure 7E white arrow).

However, the length of

filopodia formed from SaOS-2 cells cultured on PDX/PHBV/HA were higher compared to 70/30 PDX/AV mat (Figure 7F). SaOS-2 mediated cell mineralization induced the formation of HA deposits on both PDX/PHBV/HA and PDX/AV mats. However, the morphology of the particles was significantly different on the two blend systems. Indeed, HA particles on PDX/PHBV/HA mats were almost spherical in shape while those on PDX/AV fibers were “rod-like” (Figures 8A and B). Furthermore, the distribution of the formed nodules was more uniform on PDX/AV fibers compared to PDX/PHBV/HA fibers. The size of the nodules formed by SaOS-2 cells during the osteogenic differentiation process varied depending on the nature of the substrate. As can be noted from Figure 9C, PDX/AV and PDX/PHBV/HA scaffolds led to the formation of HA


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particles of sizes 250 nm vs 1 m respectively. This is an important finding since a crucial requirement for STE scaffolds is that they should induce mineralization of HA without obstructing the pores of the scaffold such that cellular infiltration is not hindered. Based on this, it can be deduced that PDX/AV is a better STE material compared to PDX/PHBV/HA. The varying conditions of HA nucleation may explain this difference in nanocrystallites morphology and size. Organic and inorganic substrates or particles play a key role in nucleation which is the first step in biomineralization which requires overcoming a free energy barrier52. They determine the crystallization process as well as the size, shape, aggregation pattern of HA53. Indeed, HA crystallites with well-defined prism shapes occurred at very low supersaturations (regime I). A compact and orderly crystalline structure was obtained with an increase in supersaturation in regime I. Aligned to the prism faces of the large crystallites were found small HA crystallites which partly showed small-angle crystallographic mismatch. Overall, this self-epitaxial nucleation led to an orderly structure. A randomly oriented and porous HA crystallite assembly resulted from enhanced surface supersaturation (in regime II). This is a result of poor structural self-epitaxial nucleation kinetics leading to randomness of the assembly. The orderly HA structure and smaller sized HA noted on PDX/AV mats suggests that cell mediated biomineralization process occurred via self-epitaxial nucleation. On the other hand, HA nucleation on PDX/PHBV/HA mats seem to follow regime II pathway leading to the formation of more random and poorly packed structures. Overall, this also implies that hard tissues resulting from biomineralization of PDX/AV electrospun mats could become tough and compact52 while porous and brittle tissues may be formed from PDX/PHBV/HA mats. Furthermore, Jiang and Lui52 successfully showed that biopolymeric molecules for instance chondroitin sulphate facilitated the alignment of a well-ordered assembly of HA crystallites. The



process occurred through nucleation and growth of daughter (or secondary) HA crystals on the surface of the existing (parent HA) crystallites. The kinetics of self-epitaxial nucleation of the daughter HA crystals were different from the parent.

by changing the kinetics of self-epitaxial nucleation of daughter (or secondary) HA crystals, which nucleate and grow on the surface of existing (parent) HA crystallites. Based on Alizarin red-S staining results, it could be concluded that 70/30 PDX/AV mats had higher potential than PDX/PHBV/HA for STE applications due to higher absorbance values and hence higher amounts of HA nodules produced during the mineralization process (Figure 9D and E). (B) In vivo biocompatibility studies: Assessment of foreign body response and angiogenesis following subcutaneous implantation of scaffolds Electrospun fiber mats were cut into 1×0.5 cm2 rectangles and implanted as three layers subcutaneously in 9 week-old male and female Wistar rats to assess their in vivo biocompatibility. The scaffolds were labelled as follows: upper layer, middle layer and lower layer. The electrospun mats and tissues surrounding the implantation sites were harvested after 2 weeks. The body weight of each animal in the study was evaluated after various time intervals (Figure S8B). A slight increase in body weight for all 5 rats was noted after 2 weeks indicating their healthy status. Upon removal of the scaffolds after 2 weeks, all of them showed signs of physical degradation with extensive tissue formation. In particular, in contrast to the other electrospun mats, the middle layer of the PDX/AV scaffold could not be separated as a result of degradation and new tissue formation. This indicates that PDX/AV mat degraded more quickly than the other mats in vivo. SEM images (Figure S9) showed that all scaffolds integrated well


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with the surrounding tissue as confirmed by the presence of tissue and cell sheets on the surface of the upper layers of all scaffolds. Indeed, the electrospun mats were interspersed with connective tissues and various types of cells. Importantly, cells and tissue infiltrated the middle layer of the scaffolds. The presence of rounded macrophage cells was noted in the upper and lower layers of all scaffolds. However, significantly more macrophages were present in the lower layer of the PDX/AV scaffold.

Figure 10: Micrographs of Masson’s Trichrome stained subcutaneous tissue surrounding (A) PDX/HA, (B) PDX/PHBV/HA and (C) PDX/AV scaffolds and quantification of (D) fibrous capsule thickness formed around the scaffolds and (E) number of blood vessels at a distance of 100 μm from the scaffolds



Any foreign material, when implanted in vivo, elicits a cascade of inflammatory mechanisms and wound-healing events that results in the encapsulation of the material in a foreign-body capsule (FBC)54. Masson’s Trichrome staining of tissue sections following removal of scaffolds depicted mild local inflammatory reactions characterized by moderate infiltration of macrophages and the absence of multinucleated giant cells (Figure 10A-C). Fibrous capsules consisting mainly of collagen fibers, fibroblasts and macrophages (Figure 10 A-C) were formed around the scaffolds. Thick and dense capsules with minimal vascular density often prevent the diffusion of analytes to and from the material thereby impeding cell growth and resulting in rejection of the material54. Therefore, the ideal TE scaffold should cause minimal inflammatory reactions when implanted in vivo with thin fibrous capsule and enhanced blood vessel formation. The quantification of the thickness of the fibrous capsules indicated that the incorporation of either PHBV or AV led to a reduction in the capsule thickness with PDX/AV resulting in the thinnest capsule formation (Figure 10D). In addition, the number of vascular structures formed around the tissues surrounding the scaffolds was significantly increased with the addition of PHBV and AV (Figure 10E). Very importantly, 46 ACS Paragon Plus Environment

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no necrotic regions were observed in the tissues surrounding all 3 scaffolds. Overall, in

vivo implantation of scaffolds demonstrated that PDX/AV mat leads to lower foreign body response compared to PDX/HA and PDX/PHBV/HA scaffolds with enhanced blood vessel formation around the latter and hence the better suitability of PDX/AV mats over the PDX/HA and PDX/PHBV/HA for TE applications. 4.0 Conclusions This exhaustive study of STE scaffolds, in which electrospun PDX/AV mats were tested in comparison to PDX/PHBV/HA against a range of cell lines involved in the skeletal tissue healing process, shows the highly promising potential of the PDX/AV mats. The scaffolds did not induce any significant inflammatory response. The differences in phase separation and hence wetting of the various blend mats led to different fibroblast-scaffold interactions, which resulted in thinner microtubules and a significantly more extensive network of actin on PDX/AV mats compared to PDX/PHBV/HA. The shape, size and abundance of HA mineralized nodules was found to depend markedly on the nature of the blend system. In particular, SaOS-2 cell-mediated biomineralization resulted in the formation of fewer random spherical microparticles on PDX/PHBV/HA mats in contrast to the abundant nano-sized “rod-like” particles noted on PDX/AV fibers. Furthermore, compared to PDX/PHBV/HA, PDX/AV mats resulted in reduced fibrous capsule thickness and enhanced blood vessel formation, as shown in in vivo biocompatibility tests. Thus, AV based PDX scaffolds are clearly more promising than PDX/PHBV/HA for STE applications.



AUTHOR INFORMATION Corresponding Authors * Dr. Nowsheen Goonoo

Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (C μ), University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany

Biomaterials, Drug Delivery and Nanotechnology Unit, Centre for Biomedical and Biomaterials Research, MSIRI Building, University of Mauritius, Réduit, Mauritius [email protected], [email protected]

* Prof. Dr. Holger Schönherr Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (C μ), University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany [email protected]

* Dr. Archana Bhaw-Luximon 48 ACS Paragon Plus Environment

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Biomaterials, Drug Delivery and Nanotechnology Unit, Centre for Biomedical and Biomaterials Research, MSIRI Building, University of Mauritius, Réduit, Mauritius [email protected], [email protected]

Funding Sources

The authors acknowledge generous financial support from the Alexander von Humboldt Foundation (Georg Forster postdoc stipend to NG), the European Research Council (ERC project ASMIDIAS, Grant no. 279202) and the University of Siegen. NG, ABL and DJ thank the Mauritius Research Council for financial support through the Nanotechnology programme, UoM Pole of Innovation for Health – MRC funded and for a PostDoctoral Fellowship to NG. NG, ABL, DJ, FG and SB thank the EU INTERREG OI / POE FEDER for financial support through the NanoMauRe project.

Acknowledgments



We thank Dr. Yvonne Voß, Dipl.-Ing. Gregor Schulte, Dipl.-Chem. Ing. Petra Frank for their support and helpful advice, as well as Dr. Ulrike Ritz (University Medical Center of the Johannes Gutenberg University Mainz, Germany) and Dr. Jürgen Schnekenburger (Biomedical Technology Center of the Medical Faculty Münster, Germany), who kindly provided the cell lines. We are also grateful to Prof. Dhanjay Jhurry (CBBR, University of Mauritius, Mauritius) for his insights into the manuscript, Jessica Andries (RIPA, GIP CYROI, Ile de La Reunion, France) and Dr. Wildriss Viranaicken (UM 134, Ile de La Reunion, France) for access to some facilities.

SUPPORTING INFORMATION Figure S1: SEM images of electrospun PDX/PHBV/HA (A) 100/0/5, (B) 90/10/5, (C) 70/30/5. Table S1: Summary of DSC results. Table S2: Summary of TGA results. Figure S2: 1H-NMR spectrum of extracted AV powder in D2O. Figure S3: MTT assay results following NIH3T3 culture on PDX/PHBV/HA and PDX/AV fibers on day 3. All measured absorbance from the blend fibers were compared with PDX/PHBV/HA (100/0/5): * p < 0.05; ** p< 0.0001 and (ns) not significant. Figure S4: Fluorescence microscopy images of RAW 264.7 cells on PDX/PHBV/HA (A) 100/0/5 (B) 90/10/5 (C) 80/20/5, (D) 70/30/5 and (E) PDX/AV 70/30. 50 ACS Paragon Plus Environment

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Figure S5: SEM image of PDX/PHBV/HA 70/30/5 showing capillary-like tube structure after 7 days. Figure S6: Cells stained with FDA on PDX/PHBV/HA (A) 100/0/5 (B) 90/10/5 (C) 80/20/5, (D) 70/30/5 and (E) PDX/AV 70/30. Figure S7:

SEM images of SaOS-2 cell-mediated mineralization after 14 days on

PDX/PHBV/HA (A) 100/0/5 (B) 90/10/5 (C) 80/20/5 (D) 70/30/5, PDX/AV (E) 70/30 fibers. Figure S8: (A) Graphical illustration of implantation procedure and (B) weight profile of the rats over the 2 weeks implantation period. Figure S9: SEM images of scaffolds removed from rats after 2 weeks.

ABBREVIATIONS AM, acemannan; AV, aloe vera; bFGF, basic fibroblast growth factor; BTE, bone tissue engineering; DSC, differential scanning calorimetry; DMEM, dulbecco’s modified Eagle’s media; ECs, endothelial cells; ECM, extracellular matrix; EDX, energy dispersive X-ray; EV, extracellular vesicles; FBC, foreign body capsule; FBS, fetal bovine serum; FDA, fluorescein diacetate; FUC; fucoidan; HA, hydroxyapatite; HFIP, 1,1,1,3,3,3-hexafluuoroisopropanol; HMDS, Hexamethyldisilazane; KCG, kappa-carrageenan; NMR, nuclear magnetic resonance; PBS, phosphate buffer solution; PCL, polycaprolactone; PDX, polydioxanone; PI, propidium iodide; PLGA, poly(lactide-co-glycolide); SBF, simulated body fluid; SEM, scanning electron microscopy; STE, skeletal tissue engineering; TGA, thermogravimetric analysis; TE, tissue engineering

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Improved multicellular response, biomimetic mineralization

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