Preclinical Alternative Model for Analysis of Porous Scaffold Biocompatibility Applicable in Bone Tissue Engineering.
Regeneration maintains or renews the original tissue architecture. In case of damage or chronic degenerative disease, fibrotic repair occurs instead of the normal regenerative process. Such a response is considered a reparative process, since the replacement tissue neither contains the original cell types nor is the original tissue architecture reestablished (Stocum, 2002).
The term regenerative medicine is often used as a synonym for tissue engineering, though regenerative medicine focuses on using stem cells to form tissues, while tissue engineering makes use of a combination of cells, biochemical and physiochemical factors, and biomaterials to improve or replace biological functions (Langer and Vacanti, 1993). Many definitions of tissue engineering include a wide range of applications, however, in practice, tissue engineering closely focuses on repairing or replacing parts of or entire tissues (cartilage, bone, blood vessels, skin, muscle, nerves, etc.) including certain mechanical and structural properties that are required for proper functioning (Musumeci et al., 2014).
Angiogenesis is the physiological process of forming new capillaries from existing vessels. It is a tiered process involving activation of the existing endothelial cells, degradation of the extracellular matrix, proliferation and migration of endothelial cells, invasion of the stroma by the surrounding cells, and remodeling the extracellular matrix. Regulation of endothelial cell survival and migration strongly depends on the interaction of endothelial cells with extracellular matrix proteins via cell adhesion molecules, and the activities of growth factors and cytokines. Angiogenesis is characteristic of regeneration of normal tissues and hence is an important factor determining the safe and successful use of biomaterials in regenerative medicine (Chavakis and Dimmeler, 2002) as perfusion of the implant is needed to provide a feasible infrastructure upon which the new tissue can mature (Anderson et al., 2011). Induction of angiogenesis and subsequent development of a vascular bed in the engineered tissue is being actively pursued through combinations of physical and chemical cues, notably through the presentation of suitable topographies and growth factors (Klagsburn and Moses, 1999; Liu et al., 2012; Kant and Coulombe, 2018).
Osteogenesis, the formation of bone, is closely connected with angiogenesis during the process of bone healing (Yu et al., 2017). One way to achieve a tissue-engineered bone tissue could be the use of an angiogenesis-promoting scaffold. Neovascularization into the implant not only supplies nutrients, oxygen, calcium, and phosphate, but also provides a transport route for the mesenchymal stem cells to facilitate bone regeneration (Stegen et al., 2015).
Scaffold that can promote osteogenesis and angiogenesis must have appropriate pore structure, biocompatibility, mechanical properties and processability to promote cell adhesion, migration, proliferation, and differentiation (Zhang et al., 2015). Chitosan is a hydrophilic biopolymer polysaccharide that induces and stimulates connective tissue rebuilding (Muzzarelli et al., 1988). It is a natural polymer with antibacterial properties that is biodegradable, biocompatible, highly abundant, and non-toxic. It can be molded into different forms (films, gels, sponges, fibers, nanoparticles, nanofibers) for diverse applications in tissue engineering (Concha et al., 2018; Poonguzhali et al., 2018). Because of the swelling of chitosan-based composites owing to water uptake during preparation, porous scaffold can be prepared by later lyophilization (Giretova et al., 2016). In bone tissue engineering, chitosan has frequently been used in combination with other bone-forming matrices such as hydroxyapatite or collagen (Tan et al., 2014). Poly-3-hydroxybutyrate (PHB) represents another natural, biodegradable and hydrophobic biopolymer that can be combined with chitosan or calcium phosphate to composites that are of interest for bone and cartilage regeneration and that may be suitable for the reconstruction of deeper defects.
The animal models play a key role in basic medical research. Advances in tissue engineering that lead to development of new therapeutic approaches are often based on in vitro experiments that are later followed by in vivo experiments. However, the use of rodents as model animals in experimental studies encounters ethical, practical and technical problems, which limit their use in certain areas of this research (Rashidi and Sottile, 2009). The avian embryo represents an easily available and cost-effective alternative model that may be used to test various biomaterials. Chicken or quail embryo development takes place outside the mother's body, obviating the need to sacrifice experimental animals or cause physical harm as is usually the case in implantation surgery. A further big advantage is that there is no need to apply for animal protocol approval for the chick/quail embryo in ovo as an experimental model as both are exempt from the horizontal legislation on the protection of animals used for scientific purposes in Europe (2010/63/EU), as well as applicable laws in the United States. The avian embryo, and especially its chorioallantoic membrane (CAM), thus provides a simple and effective alternative model to assess the biocompatibility of potential new bone implants (Kang et al., 2018; Tomco et al., 2017; Magnaudeix et al., 2016; Steffens et al., 2009) and can be considered a 3R method.
The CAM is used in many areas of research, including investigating molecules regulating the formation of blood vessels for use in the treatment of chronic inflammation, tumors, healing of wounds and fractures, drug delivery, and toxicological analysis (Hazel, 2003; Eun and Koh, 2004; Ozcetin et al., 2013; Pandit et al., 2017; Ribatti, 2016). The CAM consists of two extra-embryonic membranes: the chorion and the allantois, which fuse together on embryonic day (ED) 4. Histologically, the CAM is comprised of three layers: the ectoderm, mesoderm, and endoderm. It serves as a respiratory and excretory organ with a non-innervated, transparent matrix and a vascular network (Tay et al., 2012). On ED8, the proliferation and differentiation of blood vessels is already well advanced, forming an extensive and easily accessible arteriovenous system composed of the umbilical arteries and veins (Djonov et al., 2000), which can serve as a surrogate blood supply for organ culture, and hence a platform for biomaterial testing (Borges et al., 2003; Moreno-Jimenez et al., 2017). Over 700 publications have used the chick embryo CAM as a model system to study angiogenesis. More rarely, the Japanese quail CAM has also been used successfully (Burikova et al., 2016). A particular benefit of the quail CAM is that the quail-derived endothelium expresses the unique marker QH1, which can be identified using a specific antibody (Pardanaud et al., 1987; Drake et al., 1997; Brown et al., 1999; Giles et al., 2005; Sedmera and McQuinn, 2008).
In recent years, the field of tissue engineering has accepted the CAM model as a useful, quick, and cheap alternative to the traditional animal models (rabbit ears, rodent skin, avascular cornea of the rabbit, cheek pouches of the hamster; Ribatti et al., 2006; Da-Lozzo et al., 2013; Vargas et al., 2013) to evaluate angiogenesis in new implants and weed out less promising scaffold candidates, thus reducing or replacing in vivo animal experiments (Falkner et al., 2004).
This study describes a new approach to detect the formation of blood vessels inside biopolymer scaffolds using the CAM of the developing avian embryo as an alternative to traditional mammalian models, which would make this model particularly attractive for the rapid biocompatibility screening of porous biomaterials for regeneration of hard tissues.
2 Materials and methods
Preparation and characterization of composite scaffold Polyhydroxybutyrate/chitosan scaffolds (PHB/CHIT) were prepared according to the method of Medvecky et al. (2014). Briefly, polyhydroxybutyrate (GoodFellow, Cambridge, England) dissolved in propylene carbonate (1% solution of PHB) and chitosan (Sigma Aldrich, middle, 1% solution in 1% acetic acid) were mixed together at a 1:1 ratio using a magnetic stirrer at 400 rpm. After 10 min of mixing, acetone (about 5 ml) was slowly added to the suspension to achieve precipitation of biopolymers. Final blends were filtered, washed with distilled water, and molded into a larger block (4x25x1 mm), which was then cut into smaller pieces with the final dimensions of 4x4x1 mm and lyophilized (Freeze dryer, ilShin Biobase Europe, Ede, The Netherlands) for 6 h (Fig. 1). Swelling of the composite samples was measured in 1.5 ml vials by immersion of porous substrates (approximately 20 mg) in 0.9% NaCl solution at 37[degrees]C up to a constant mass. Soaking was done in triplicate and swelling was evaluated as the ratio of wet weight to dry weight. The microstructure of the scaffolds was observed by scanning electron microscopy (FE SEM JEOL7000). The phase analysis of the blend was evaluated by X-ray powder diffraction analysis (XRD, Philips X Pert Pro).
Cytotoxicity evaluation was carried out using MC3T3E1 mouse preosteoblasts (ECACC, Salisbury, UK) according to EN ISO 10993-5:2009. Cells were cultured in culture flasks in MEM (minimum essential medium) with Earl's balanced salts, 2 mM L-glutamine (SAFC Biosciences, Hampshire, UK), 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), and ATB-antimycotic (penicillin, streptomycin, amphotericin) solution (Sigma-Aldrich, St. Louis, MO, USA) at 37[degrees]C in 5% C[O.sub.2] at 95% humidity. After the cells reached about 80% confluence, they were harvested using 0.25% trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA). Scaffold samples were sterilized in an autoclave at 121[degrees]C. Cell viability in the presence of scaffold samples was examined using the MTS test (Cell titer 96 aqueous one solution cell proliferation assay, Promega, Madison, WI, USA). The sterilized scaffolds ([empty set] 6 mm, thickness 1 mm, n = 5) were placed into the plate (treated 96-well tissue culture plate, cellGrade Brand, Wertheim, Germany), seeded with 1.0 x [10.sup.4] MC3T3E1 mouse preosteoblasts in 200 [micro]l complete osteogenic medium alpha modification MEM with 50 [micro]g/ml ascorbic acid, 50 nM dexamethasone, and 10 mM [beta]-glycerophosphate (Sigma Aldrich, St. Louis, MO, USA) and cultured at 37[degrees]C in 5% C[O.sub.2] at 95% humidity. Cell viability in the presence of scaffolds was evaluated 2 and 10 days after cell seeding by measuring formazan concentrations produced by metabolically active cells in the culture medium using a UV-VIS spectrophotometer (Shimadzu, Griesheim, Germany) at a wavelength of 490 nm. The mean measured absorbance of medium from wells with cell-seeded substrates was compared with the absorbance of medium from scaffold-free wells (negative control, n = 5). The pure complete culture medium was used as a blank.
The attachment and morphology of cells were visualized by live/dead fluorescence staining using fluorescein diacetate/propidium iodide solution (green, live cells and red, dead cells) after 48 h of cell cultivation in the presence of scaffolds by an inverted optical fluorescence microscope (Leica DM IL LED, blue filter).
Chick CAM ex ovo model for microscopical evaluation
Avian embryos used in this study are exempt from Directive 2010/63/EU. Fertilized chicken hybrid eggs (Ross 308; n = 14) were purchased from the chicken farm of the Institute of Molecular Genetics (Kolec, Czech Republic) and delivered via courier in a temperature-controlled manner to ensure egg viability and quality. The eggs were incubated horizontally in a forced-draft constant-humidity incubator at 37.5[degrees]C and 60% relative humidity. At ED3 the eggshell was disinfected with 70% ethanol, then cracked and the egg content with chick embryo was carefully transferred into a hexagonal plastic weighing boat stored in a Petri dish partially filled with sterilized distillated water to maintain humidity, and incubated until ED6 in a still draft incubator (37.5[degrees]C, 70% relative humidity, without rocking; McQuinn et al., 2007).
On ED6, a piece (2x2x1 mm) of the sterilized porous scaffold (CHIT/PHB) was gently placed on the chorioallantoic membrane ex ovo using suture tying forceps.
For visual evaluation of vascular density and video blood flow observation into/outside the scaffold we used a stereomicroscope (Leica MZ125) fitted with a DSLR camera (D7000, Nikon, Tokyo, Japan) for video documentation on ED10. Blood contrast was enhanced using a green interference filter inserted into a KLD250 halogen light source. Ultrasound biomicroscopy examination of implants was performed ex ovo on a high-resolution imaging system Vevo 770 (VisualSonics, Toronto, Canada) using B-Mode imaging (scanhead #708), which allows the acquisition of two-dimensional images of the vessels inside of scaffolds as well as blood flow monitoring in a temperature-controlled setup (McQuinn et al., 2007).
Quail CAM in ovo model for evaluation of angiogenesis and biocompatibility
For implantation of scaffolds on the quail CAM in ovo, we used a method modified from Ribatti et al. (2006). Fertilized Japanese quail (Coturnix japonica) eggs (n = 10) were purchased from the animal farm (Kosice, Slovakia) and delivered via courier in a temperature-controlled manner to ensure egg viability and quality. The eggs were incubated with blunt end up in a forced-draft constant-humidity incubator at 37.5[degrees]C and 60% relative humidity with continuous rocking. At ED3 the eggs were windowed on the blunt end, and the inner shell membrane (membrana papyracea) was carefully removed. The windows were closed using insulation tape and returned to a still draft incubator.
On ED5, a sterilized porous scaffold (CHIT/PHB) was gently placed on the chorioallantoic membrane, and the opening was re-sealed with the insulation tape. On ED10, Dent's solution was applied directly onto the CAM with the scaffold and surrounding vessels for 10 min at room temperature. Subsequently, the implant was removed and processed for evidence of angiogenesis and biocompatibility with histological techniques.
Scanning electron microscopy (SEM)
SEM analysis was used to investigate whether CAM microvilli grow into the pores of the scaffold. This would evidence biocompatibility of the tested biomaterial. In ovo implants at ED10 were fixed with 1% glutaraldehyde and 2% formaldehyde in PBS for 24 h at 4[degrees]C, rinsed three times with PBS, and then post-fixed in 1% osmium tetroxide. After further rinsing, the specimens were dehydrated with ethanol series, critical point dried with CO2, and mounted on aluminum stubs. After sputter coating with gold, they were observed under a Bruker scanning electron microscope.
The monitoring of the implant reaction and biocompatibility with the surrounding CAM was carried out using routine H&E/Alcian Blue staining, followed by scanning of the slides using a 10x objective on an Olympus slide scanner.
The presence of myofibroblasts and macrophages ([alpha]-SMA, collagen I and CD68, respectively) as well as proliferative activity of the cells within the implant (phosphohistone-3, Anti-H3S10p) was evaluated with immunohistochemical staining of chick embryos. Deparaffinized sections were blocked in normal goat serum (1:10) and in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton-X (Sigma-Aldrich, St. Louis, USA) for 60 min at RT. For staining with CD68 antibody, collagen I, and anti-H3S10p, antigen retrieval was performed with 10 mM citrate buffer (pH 6) in a microwave oven (750 W) for two cycles of 5 min each prior to immunostaining. Primary antibodies (monoclonal mouse antibody [alpha]-SMA, 1:800, Sigma-Aldrich # A2547; polyclonal rabbit anti-mouse antibody collagen type I, 1:500, MDbiosciences, Oakdale, USA; monoclonal mouse antibody CD68, 1:100, Abcam ab955, Cambridge, UK or polyclonal rabbit antibody anti-H3S10p, 1:100, Millipore, CA, USA) were applied overnight at +4[degrees]C. Negative controls were obtained by omission of the primary antibody. The sections were then washed in PBS, and TRITC-conjugated goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody for collagen type I (1:200, Jackson ImmunoResearch Laboratory, West Grove, PA, USA) was applied for 90 min in the dark at RT. Together with the secondary antibody, wheat germ agglutinin (WGA) coupled with the Alexa 488 dye was applied at 1:100 concentration to detect fibrous extracellular matrix including collagen (Benes et al., 2011). After washing in PBS, the nuclei were counterstained with Hoechst 33258 (1:100,000 diluted in 0.1% Triton-X in distilled water, Sigma-Aldrich, St. Louis, MO, USA). The sections were washed with distilled water and dehydrated in ethanol series, cleared in xylene, and mounted in DEPEX (Electron Microscopy Sciences, Hatfield, PA, USA).
The presence of hemangioblasts and endothelial cells was investigated with QH1 staining. Deparaffinized sections of ten quail embryos with implants were blocked in normal goat serum (1:10) and in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton-X (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at RT. QH1 (monoclonal mouse antibody, 1:1000, DSHB, Iowa, USA) was then applied overnight at +4[degrees]C. Negative controls were obtained by omission of the primary antibody. The sections were washed in PBS and TRITC-conjugated goat anti-mouse secondary antibody (JacksonImmunoResearch Laboratory, West Grove, PA, USA) was applied for 4 h in the dark at RT. After washing in PBS, the nuclei were counterstained with Hoechst 33258 (1:100,000 diluted in 0.1% Triton-X in distilled water, Sigma-Aldrich, St. Louis, MO, USA). The sections were washed with distilled water and dehydrated in ethanol series, cleared in xylene, and mounted in permanent medium.
Fluorescently stained sections were examined first on a wide-field epifluorescence microscope (Olympus BX51) and then documented on a confocal microscope Olympus FV-1000 BX61 (Olympus, Tokyo, Japan). Images were assembled and labeled using Adobe Photoshop (version 8.0) using Wiley standards for digital image manipulation. Brightness and contrast were adjusted using the "Adjust Levels" command on the entire image, and "Unsharp Mask" filter was used where necessary to enhance sharpness. No other image manipulations were performed.
CAM tissue remodeling in relation to the implanted scaffold was further analyzed using picrosirius red staining according to Junqueira et al. (1979). This specific staining of extracellular collagen fiber deposition was combined with immunohistochemical staining of WGA coupled with the Alexa 488 dye (1:100), and the nuclei were counterstained with Hoechst 33258 (1:100,000 diluted in 0.1% Triton-X in distilled water). For histological and immunofluorescent studies, we used 4 sections per slide, 2 slides per sample.
3.1 Properties, microstructure and in vitro cytotoxicity of composite scaffolds
A macroporous microstructure with high fractions of spherical pores, which did not exceed 80 [micro]m diameter, and irregular macropores, with diameters up to 150 [micro]m, was observed in PHB/CHIT scaffolds (Fig. 1) by SEM. These pore sizes appear appropriate for migration of cells into the inner structure of the scaffolds to form specific tissues. No clearly distinguishable biopolymer fibers were visible in the detailed micrograph, which demonstrates uniform and homogeneous distribution of both biopolymers in the blend.
Dimensions of individual fibers have to be sufficient for crystallization into coherent regions, which was demonstrated by the XRD analysis (Fig. 2). The strong lines from reflections of (020), (110), (101), and (121) PHB planes verify a higher fraction of the crystalline PHB component in the PHB/CHIT blend contrary to the amorphous character of chitosan. A significant reduction of the average molecular mass of chitosan from 360 kDa to about 41 kDa was found after precipitation of biopolymers, which is probably the reason for low chitosan crystallinity.
A fast increase in scaffold mass was observed after the first 2 h of soaking when the mass increment achieved about 90% of the final water uptake. Constant mass of samples was reached after 2 days of swelling at which the dry mass of the biopolymer scaffold had more than doubled. This confirmed a highly hydrophilic character of the blend, which supports the adsorption of polar molecules (like nutrients, etc.) from the surrounding medium including body fluids.
Formazan production of preosteoblasts in contact with the scaffolds for 2 and 10 days of culture (results not shown) indicated an increase in the number of viable cells (about 7-fold), which demonstrates that the scaffolds support cell growth. The relative viability of cells increased from 50% after 2 days of culture to around 80% of the negative control after 10 days of culture, which confirms a non-cytotoxic behavior of biopolymer scaffolds (EN ISO 10993-5; Fig. 3). Images from live/dead fluorescence staining (Fig. 4) documented good adherence of cells to pore walls of scaffolds 2 days after seeding. The live cells' visible filopodia copied the pore walls and only few dead cells were observed (stained red; panel A, B of Fig. 4).
3.2 Incorporation of scaffold into the CAM
Five days after placing the PHB/CHIT scaffold on the CAM, it was well incorporated. Hyperplasia of the CAM tissue under the scaffold was observed in all cases. Epithelial cells from the CAM ectoderm were observed to proliferate and move into the PHB/CHIT component, forming fusion boundaries between the scaffold and CAM tissues, suggesting good biocompatibility and bioactivity of this biomaterial. Newly formed CAM tissue was observed also within the PHB/CHIT scaffold as the formation of CAM villi. These cells expressed smooth muscle actin, suggesting they were myofibroblasts. They were localized only in some areas around the implant. However, there was no excessive production of collagen I or extracellular matrix as revealed by WGA or picrosirius red staining (Fig. 5). CD68 staining did not identify the presence of macrophages.
3.3 Angiogenesis within the scaffolds on the CAM
Endothelial cell migration and sprouting are the most important steps of angiogenesis. We observed vessels leading to and from the implant on the surface of the CAM (Fig. 1), suggesting its vascularization after 5 days of implantation. This was confirmed by high-resolution videomicroscopy (Video S2 (2)) and ultrasound biomicroscopy (Video S3 (3)). The presence of endothelial precursors and vessels inside the implant was confirmed by positive QH1 staining (Fig. 6) on scaffolds implanted into the quail chorioallantoic membrane.
Cell adhesion to an implant and cell activity are influenced by properties of the implant surface such as surface tension, roughness, etc. (Wang et al., 2005). Chitosan provides a reservoir for the release of bioactive substances and also acts as a scaffold that fisamenable to colonization by cells (Barreto et al., 2016; Medvecky et al., 2014).
Polyhydroxybutyrate porous scaffolds in combination with biphasic calcium phosphate or chitosan are known to stimulate proliferation of fibroblasts and osteoblasts without inducing a proinflammatory response (Cool et al., 2007; Veleirinho et al., 2012; Tai et al., 2014). The average molecular weight of chitosan decreases after precipitation in propylene carbonate solution, but this does not appear to have negative effects as Giretova et al. (2016) found that lysozyme-degraded scaffolds containing a large fraction of low molecular weight chitosan (LMWC) were not cytotoxic even after 10 days of cultivation.
Previous studies have reported that various versions of chitosan-based scaffolds may support angiogenesis for different applications (Deng et al., 2010; Wang et al., 2012; Linn et al., 2003; Agarwal et al., 2016; Shahzadi et al., 2016); however only few studies have investigated this for the field of bone tissue engineering and these were only able to show evidence of angiogenesis on the scaffold surface (Pan et al., 2018; Ahmadi et al., 2010) or found that chitosan-acetate may actually inhibit angiogenesis (Shah et al., 2014). Others have attempted to enhance angiogenesis by loading chitosan scaffolds with recombinant vascular endothelial growth factor (VEGF) (Linn et al., 2003) or expressing VEGF with an adenoviral vector (Koc et al., 2014).
We here show clear evidence of blood vessel ingrowth into unseeded PHB/CHIT scaffolds without performing in vivo testing on animals and without expression or addition of VEGF. Using the QH1 antibody, we observed the presence of individual endothelial cells as well as vessels inside the porous biomaterial. However, there are also some QH1-positive cells not connected to the vessels. It is thus possible that hemangioblasts migrate into the implant and vasculogenesis takes place together with angiogenesis. This hypothesis would have to be tested by another experimental study.
We found that the chitosan implant surface has fibrotic effects, probably because the implant surface directly activates quiescent myofibroblasts to differentiate into myofibroblasts by providing a mechanical stimulus. Myofibroblasts are characterized by an excessive production of collagen (Majd et al., 2015). Using WGA labeling of extracellular matrix, which was found to be comparable with the established picrosirius red staining, we found that the fibrous layer was present around the implant outside of the layer of [alpha]-smooth muscle actin ([alpha]-SMA) positive myofibroblasts (Fig. 4; Emde et al., 2014).
We did not find evidence of induction of a non-specific inflammatory reaction by the implant as we did not find an influx of CD68 positive macrophages. This may be because we put the implant on the CAM early during development (ED5), at a time when the immune system is still immature. Inflammatory angiogenesis, in which infiltrating macrophages are the source of angiogenic factors, has to be distinguished from direct angiogenic activity of the tested biomaterial with histological analysis and immunohistochemical staining for specific cell populations. It would be interesting to extend our studies also into the later period (Baiguera et al., 2012) to monitor myofibroblasts as they become apoptotic or fibrosis develops, and to observe the influx of macrophages as the immune system develops. However, in some countries protection of the avian embryo starts at ED12-14 when the nervous system develops sensory synapses and two thirds of gestation has elapsed.
The CAM was previously used as an in ovo method for evaluating the tissue response to various biomaterials, including composites based on collagen (Vargas et al., 2013), Elvax 40 (ethelyne-vinyl acetate copolymer; Langer and Folkman, 1976), hydron (poly-2-hydroxyethyl-methacrylate polymer; HydroMed; Ribatti et al., 1996), PCL (polycaprolactone; Singh et al., 2012), and matrix hydrogels (Fercana et al., 2017). This is a rather low-tech method, which makes it possible to continuously monitor angiogenesis, to easily and quickly obtain results, and to evaluate them in a relatively short time period. The response of the CAM to a biomaterial is similar to that of the mammalian animal model. However, in contrast to the mammalian model, it allows continuous monitoring, which makes it very attractive for rapid in ovo/ ex ovo angiogenesis evaluation (Magnaudeix et al., 2016; Jin et al., 2016). Angiogenesis in the CAM model is usually quantified by assessing the number of new vessels oriented towards the implant (Ribatti et al., 2006; Strick et al., 1991). This method is rather subjective and only considers angiogenesis outside or on the surface of the scaffold. The focus of our study was primarily to identify the presence of blood vessels in the internal structure of the scaffold.
Our results showed that the PHB/CHIT scaffold has satisfactory angiogenic properties and induces formation of CAM villi penetrating the pores of the implant. One can easily quantify the number of blood vessels in the vicinity of the implant, perform topographical and structural assessment using confocal or electron microscopy, and examine the cellular changes within the structure by immunohistochemistry.
The CAM model represents an intermediate step for testing biomaterial between the simple, in vitro model and the complex in vivo mammalian animal model (Valdes et al., 2002). Thus, it can reduce and replace animal experiments in the field of bone regenerative medicine.
Original porous PHB/CHIT biomaterial designed for bone regeneration was tested for the first time with a short-term CAM assay. Previous studies monitored the pro-angiogenic properties and biocompatibility only on the surface of similar porous scaffolds based on chitosan. In this study, the methods were focused on monitoring of angiogenesis and biocompatibility inside of the scaffold, which provides more complex information for the qualitative assessment of the tested biomaterial. The methods allow observation of the surrounding CAM tissue reaction, presence of cells in the pores of the scaffold, and the comparison of vessels growing toward the implant with their actual presence inside it. The presence of hemangioblasts and endothelial cells inside the scaffold could be shown. It remains to be determined whether angiogenesis or vasculogenesis is involved. Myofibroblasts were found around the implant, but without excessive collagen deposition. This study confirmed that the CAM assay is a rapid, cost-effective and simple method to test and optimize new scaffolds before their use on larger experimental animals.
Conflict of interest
There is no conflict of interest.
The present study was supported in part by the Grant Agency of Ministry of the Education, Science, Research and Sport of the Slovak Republic no. VEGA-1/0046/16; Slovak Research and Development Agency under the contract no. APVV-17-0110; Ministry of Education, Youth and Sports of the Czech Republic PROGRESS-Q38, Charles University SVV Graduate Student Training Program; Ministry of Health AZV CR, 15-32497A, 1728265A; Czech Academy of Sciences RVO: 67985823, INTERCOST LTC17023, and LM2015062 Czech-BioImaging CZ.02. 1.01/0.0/0.0/16_013/0001775 Modernization and support of research activities of the national infrastructure for biological and medical imaging Czech-BioImaging funded by OP RDE.
The authors would like to thank Blanka Topinkova and Mariana Saulicova from the Institute of Anatomy, Charles University in Prague, Czech Republic and the Institute of Anatomy, University of Veterinary Medicine and Pharmacy in Kosice, Slovak Republic, for their laboratory facilities.
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(2) In situ blood flow from the PHB/CHIT implanted scaffold including the vessels: doi:10.14573/altex.1807241s2
(3) Blood flow inside of PHB/CHIT implanted scaffold by VeVo ultrasound system: doi:10.14573/altex.1807241s3
Eva Petrovova , Maria Giretova , Alena Kvasilova , Oldrich Benada , Jan Danko , Lubomir Medvecky   and David Sedmera [3,5]
 Institute of Anatomy, University of Veterinary Medicine and Pharmacy, Kosice, Slovak Republic;  Institute of Materials Research, The Slovak Academy of Sciences, Kosice, Slovak Republic;  Institute of Anatomy, Charles University, Prague, Czech Republic;  Institute of Microbiology, The Czech Academy of Sciences, Prague, Czech Republic;  Institute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic
Received July 24, 2018; Accepted November 12, 2018; Epub November 23, 2018; [c] The Authors, 2018.
Correspondence: Eva Petrovova, DVM, PhD; Institute of Anatomy, University of Veterinary Medicine and Pharmacy, Komenskeho 73, 041 81 Kosice, Slovak Republic
Caption: Fig. 1: Structure of PHB/CHIT scaffold and evidence of CAM blood vessels converging toward the implanted scaffold
(A) Macrostructure of PHB/CHIT scaffold, scale bar: 1 mm. (B) Ultrastructure of PHB/ CHIT scaffold with pores (SEM), scale bar: 200 [micro]m. (C) Macroscopic evidence of CAM blood vessels converging toward the implanted PHB/CHIT scaffold (arrows) on ED10; scale bar: 1 mm; 42 biological replicates (chick), 30 biological replicates (quail), for confirmatory images see (1).
Caption: Fig. 2: XRD patterns of PHB and PHB/CHIT blend Biological replicates (4).
Caption: Fig. 3: Proliferation of osteoblasts on PHB/CHIT scaffolds in relation to negative control after 2 and 10 days of cell cultivation
Biological replicates (6), p < 0.01 (one-way ANOVA, [alpha] = 0.05, StatMost statistical software).
Caption: Fig. 4: Live/dead (arrows) fluorescence staining of cells growing on scaffolds after 2 days of cultivation
Biological replicates (9), technical replicates (45), for confirmatory images see (3). Because of the porous structure of the material and nonstandard conditions for observation of cells by optical microscopy (especially flat surface), full images were not sharpened.
Caption: Fig. 5: Immunohistochemical analysis of the implant after 5 days in ovo
(A) Histology of the implant after 5 days in ovo. The formation of CAM villi growing into the implanted PHB/CHIT scaffold was observed using SEM as a comparison with histology (3D); scale bar: 400 [micro]m. (B) Confirmation of CAM villi presence with H-E/Alcian blue staining; ET, ectoderm; M, mesoderm; ED, endoderm; scale bar: 100 [micro]m. (C) Evidence of CAM tissue and proliferating cells within implanted scaffold; scale bar: 100 [micro]m. (D) The presence of myofibroblasts on a border between scaffold and surrounding CAM tissue; scale bar: 100 [micro]m. (E, F) Collagen was localized around implant without its excessive deposition; scale bar: 200 [micro]m; biological replicates (15), technical replicates (720), for confirmatory images see (1).
Caption: Fig. 6: Evidence of endothelial cells, hemangioblasts, and blood vessels inside the implanted scaffold
(A) QH1 positive endothelial cells; scale bar: 200 [micro]m.
(B) Blood vessels (asterisk) and hemangioblasts are present inside of implanted scaffold; scale bar: 100 [micro]m; biological replicates (9), technical replicates (72), for confirmatory images see (1).
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|Title Annotation:||Research Article|
|Author:||Petrovova, Eva; Giretova, Maria; Kvasilova, Alena; Benada, Oldrich; Danko, Jan; Medvecky, Lubomir; S|
|Publication:||ALTEX: Alternatives to Animal Experimentation|
|Article Type:||Technical report|
|Date:||Jan 1, 2019|
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