3.1 Scaffolds
The appearance of the freeze-gelled scaffolds was of porous white sponges (Figure 1).
Scaffolds of disc shape of 5mm diameter and 2.5 mm thickness approximately were produced. These scaffolds exhibited an average weight of 6.47 ± 0.076 mg (SD= 1.014). Concentrations of heparin loading is given in Table 1.
Sample Name
|
Matrix Composition
|
Heparin loading concentration (mg/mL)
|
MMW
|
Chitosan MMW
|
-
|
M0
|
Chitosan MMW + Hydroxyapatite
|
-
|
M0.5
|
0.5
|
M1
|
1
|
M2
|
2
|
M5
|
5
|
Table 1. Materials with variable heparin concentrations.
3.2 SEM characterization
Morphology of the freeze-gelled scaffolds was observed by SEM. The physical structure confirmed interconnected porosity and the presence of embedded hydroxyapatite crystals distributed throughout the polymer matrix were also visible. As observed in Figure 2, neither the process of heparin loading by immersion in aqueous solution nor the concentration of heparin loaded affected the structure of the scaffold, as no visible changes were observed among scaffolds with different concentrations.
The notable differences were spotted on the porosity and distribution according to the side of the disc-shaped scaffold. Figure 3 presents images from the scaffold loaded with 0.5 mg/mL for illustration purposes, all the scaffolds exhibited a similar behaviour. Chitosan-rich side was found on the top side of the scaffold and was characterised for a porosity ranging from 90 to 195 μm, Figure 3 (A). A smoother surface with scattered hydroxyapatite particles could be seen. Lateral images of the scaffold, Figure 3 (B), show a porosity distribution from 51.61 to 392.63 μm, with an average of 182.83 μm. Hydroxyapatite is evenly embedded throughout the chitosan matrix of this side. The bottom face of the scaffold exhibited the hydroxyapatite-rich side, Figure 3 (C), a more packed appearance can be observed, with an average pore size of 40 μm. The entire surface of the polymer matrix appears to be covered by the hydroxyapatite particles.
This gradient of hydroxyapatite from top to bottom originated during freezing part of the scaffold formation process. Hydroxyapatite surface charge usually is slightly negative, due the presence of hydroxy and phosphate groups on its lattice surface. However, this charge can change with pH 36. When hydroxyapatite becomes in contact with an acidic solution, there is a change on its surface charge due to the adsorption of H+ onto the hydroxyl ions and the protonation of the phosphate groups of the surface 37. Despite this change on its surface, it has affinity towards chitosan due to its positive charge, which also prevents the precipitation and keeps molecules dispersed within the chitosan matrix. Additionally, hydroxyapatite is not soluble in the solution in which chitosan was dissolved, therefore, this chitosan solution is used to form a matrix in which the phosphate ions may be suspended. During the process of freeze-gelation pores were formed by removing the solvent while the frozen shape was retained. The freezing part of the process was kept at a relatively slower rate to, giving sufficient time for the crystals to form and generate pores within the chitosan matrix. Therefore, during this process, an amount of the suspended hydroxyapatite settled at the bottom of the container, while the rest was kept in suspension and could be observed homogeneously dispersed in rest of the scaffold.
Hydroxyapatite rich side of the scaffold allows to exploit its’ bioactive property. Since hydroxyapatite is often used as coating to induce bioactivity on orthopaedic implants, mainly because of the interaction of the osteoblasts around the bioactive surface, which promotes the formation of new mineralised bone matrix.
Pore size represents a very important parameter for designing any scaffold in tissue engineering. It influences the cellular activity in terms of attachment, matrix deposition and differentiation. It seems to exist a general agreement within the field of bone tissue engineering to consider that the optimal pore size for bone tissue scaffolds is between 100 and 500 μm, to have good cell adhesion and proliferation, and vascular ingrowth38,39.
3.3 FTIR Characterization
Chemical composition was characterised by ATR-FTIR spectroscopy. Top and bottom surfaces of the disc-shaped scaffolds were analysed, showing the formation of a functionalized scaffold, with a chitosan-rich surface and a hydroxyapatite-rich surface, as described in the previous section. Figure 4 shows the resulting spectra comparing both surfaces of the scaffold and the precursors used (chitosan and hydroxyapatite). FTIR spectra confirmed the composition of the scaffold and, in accordance with the SEM results, show the clear difference between the two surfaces. For comparison purposes hydroxyapatite spectrum (C) was placed right below the bottom surface spectrum (D), the same case with chitosan (A) and the top surface (B).
Hydroxyapatite and the bottom surface showed well-defined phosphate (PO4) peaks at 564 cm-1 and 1018 cm-1, corresponding to phosphate n4 and n3 stretching modes, respectively. Likewise, hydroxyl (OH) stretch peak was observed at 3571cm-1 for HA spectra 40.
Chitosan was present on the top surface and the corresponding spectra showed a band for alkyl (CH2, CH3) bending at 1375-1415 cm-1, a small hump between 1586 and 1650 cm-1 corresponding to the CO stretching from Amide I, and, the peaks of CH2, CH3 stretch near 2850-2915 cm-1 24,41. These spectra also showed a band between 1025-1060 cm-1 assigned to C-O-C stretching and a broad peak at 3555 cm-1, which may be attributed to a combination of OH and NH, stretching vibrations 24,41.
3.4 Heparin Loading determination
Toluidine Blue assay allowed the quantitative determination of the heparin content as well as its qualitative distribution on the scaffold. The distribution throughout the scaffold was uniform, showing the change of colour from blue to purple along the chitosan matrix. The change of colour in the Toluidine Blue solution is immediate after the immersion of the scaffolds, with gradual differences in the shades of blue and purple according to the heparin concentration, the colours of the graph intend to mimic the shades observed. From Figure 5 (A to E), it is possible to notice the difference between the sample without heparin which exhibits a completely blue surface, and the rest of the samples with heparin, which can be seen as purple surfaces with some scattered blue spots. However, the purple shade in the heparinized scaffolds did not give much information about the concentration of each sample, as no apparent difference among scaffolds was notable, regardless of the concentration.
Regarding the quantification of the heparin, Figure 6 presents a graph showing the amount of heparin released throughout the different intervals. It is possible to observe that the amount of heparin detected on every scaffold varied according to the loading concentration where they were immersed. By using these data, we obtained the total sum of all the results giving us an estimated total concentration of heparin per scaffold, showed in Table 2. Average concentrations from 28.14 μg to 315.89 μg per scaffold were achieved.
Loading Concentration
mg/ mL
|
Approximate Content
µg /scaffold
|
% release within the first 1.5 h
|
0.5
|
28.14
|
44.68
|
1
|
50.14
|
57.06
|
2
|
138.85
|
82
|
5
|
315.89
|
90.38
|
Table 2. Average total content per scaffold.
The loading of heparin onto chitosan is feasible due to the presence of positive charge (amine groups) in the polymer matrix and anionic charge (sulphate and carboxyl groups) of heparin. However, it is important to highlight that during this study most of the heparin was released within the first 1.5 hours of immersion in water as shown in Figure 6. The higher the loading concentration of the scaffold the higher the percentage of heparin content release within the first 1.5 hours, with the maximum concentration losing the 90% of the heparin content within this time. Gümüșderelioǧlu & Aday (2011) reported a fast release with their functionalized chitosan scaffold where heparin had been bonded electrostatically 25. The functionalization of chitosan scaffolds with heparin was caused by the presence of positive charge (amine groups) in the polymer matrix and anionic charge (sulphate and carboxyl groups) of heparin. Therefore, the DD of the chitosan plays an important role on heparin loading, our matrix was prepared with a chitosan with DD ≥ 90%, thus, presenting enough amino groups for the heparin interaction. The abovementioned authors, however, report a release of at least 50% within the first 10 days of exposure to cell culture process. This means that the addition of hydroxyapatite, causes the electrostatic interaction between chitosan and heparin to be weaker, since the distribution of hydroxyapatite throughout all chitosan matrix hinders their interaction. Additionally, hydroxyapatite exhibits a slightly negative charge due the presence of phosphate and hydroxyl groups. This negative charge causes a repulsion of between these two components during their interaction in the matrix. This explains the faster release when more heparin was presented. Covalent bonding of heparin would represent an option worth to explore, as well as the response of this bonding and the addition of hydroxyapatite.
It is also important to note that the presence (or release) of heparin from the scaffolds loaded with the lowest loading concentration, was more constant over time. This is notable by day 5, when they still show the presence of more than 20% of the total of heparin. Meaning that lower loading concentrations allow a better interaction of the heparin solution with the chitosan/hydroxyapatite matrix and provide a true sustained release for a longer period.
3.5 In-vitro Degradation
The purpose of exposing the material to the action of enzyme was to mimic the degradation it undergoes during implantation, lysozyme is present in various human fluids in varied concentrations 42. For comparison purposes, scaffolds of just chitosan medium molecular weight (MMW) were prepared via freeze- gelation methodology.
Figure 7 shows the variations of the scaffold structure over time under the immersion in the PBS + Lysozyme. It is important to mention that though the presence of heparin and the loading methodology did not affect the scaffold structure as mentioned on section 3.2, when comparing pure chitosan scaffolds with hydroxyapatite scaffolds, the latter present closed porosity, i. e. the porosity looks tighter and the pore size smaller, which means that the presence of hydroxyapatite closes down the pore structure of the chitosan matrix. This is mainly formed when preparing the solution for the scaffolds, as the addition of hydroxyapatite into the chitosan solution, results in a thicker solution due to the attraction between chitosan and hydroxyapatite, as described in section 3.2. This solution results denser zones undergoing freeze-gelation process, and consequently smaller pore sizes. Additionally, during freeze-gelation process, there is some remelting of the solution, caused by the exothermic neutralization with the sodium hydroxide solution for the gelation phase. This slight remelting creates different zones within the frozen solution, some more compact than the others, generating a difference on their porosity 43.
Regarding the influence of degradation media, it was observed that for day 7 there was precipitation of salts all over the scaffolds with hydroxyapatite, particularly for the scaffold with heparin. By day 14 and 21 a similar trend was observed with all the scaffolds, salts precipitation on hydroxyapatite/scaffolds-surface, but all of them presented slightly open porosity, which may be due to the degradation of chitosan matrix. This is evident from the graph presented in Figure 8, showing the percentage of dry weight remaining after degradation protocol.
Generally, immediately after immersion in liquids, chitosan hydrogels tend to swell and retain water, which can explain little or non-existent weight loss of the first day of immersion. However, with time the swelled structure provides a higher porosity and surface area that favours lysozyme degradation of the structure and loss of the weight and integrity of the scaffold. It was observed in this study only for chitosan scaffold, the weight loss was immediate since lysozyme degrades chitosan by hydrolysing its glucosamine bonds. But in this case, chitosan matrix degradation was affected by the presence of hydroxyapatite, which hampers lysozyme interaction with chitosan. Lysozyme adsorbed onto hydroxyapatite surfaces, favoured by the interactions of lysozyme functional groups and hydroxyapatite phosphate groups 44. Furthermore, chitosan matrix degradation depends on pH, and the presence of heparin decreases protonation of chitosan amino groups due to its complexation with the negative charged heparin-functional groups, neutralizing pH with time 45.
Therefore, the behaviour of the scaffolds could be due to the deposition of lysozyme on hydroxyapatite, and the presence of heparin holding up chitosan degradation. In addition, weight loss and a slight yielding of chitosan structure was also observed after 21 days of exposure.
3.6 Cytotoxicity
Cell viability evaluation determines the ability of the material to maintain the cells alive within the scaffold during a certain period. The scaffold should provide a suitable environment in terms of attachment and space for their migration. Chitosan and hydroxyapatite scaffolds are already known to offer the appropriate biocompatible characteristics for cell survival 26. The evaluation of cell viability was performed by means of an Alamar Blue assay. In this method, the measurement of the absorbance in the cell media provides an estimation of living cells, given that living cells reduce resazurin (non-fluorescent, low absorbance) to resorufin (highly fluorescent, high absorbance).
The metabolic activity of U2OS cells on the scaffolds is presented in Figure 9. The cells were evaluated for 14 days, with readings taken on the 1st, 4th, 9th and 14th day. According to these results, regardless of the heparin content, cell activity remains constant throughout the entire evaluation. Proving cell attachment, during the first day, and cell survival, until the last day of the assay. It can be observed that for the scaffolds without heparin, the proliferation of cells was greater in time (higher metabolic activity) when compared to the behaviour observed in the scaffolds with different heparin concentrations.
Furthermore, the heparin concentration hinders cells proliferation. Therefore, we observe that higher heparin contents result in less metabolic activity. Our scaffolds present a fast desorption of heparin, as mentioned in the previous section, which does not allow heparin to interact with fibronectin and other attachment factors 25, causing the low proliferation profile in these results. Similar results were presented by Gümüșderelioǧlu & Aday (2011), scaffolds with electrostatically loaded heparin reporting no particular proliferation during their study, however, showing important cell differentiation results 25.
3.7 Angiogenic activity ex-ovo CAM Assay
The CAM functions as a gas exchanger, and a disposer for the chick embryo’s waste. It also plays part in the mineral transport for the bone development of the embryo. These activities are mainly due to its location and its highly vascularized nature 46. Ribatti (2017) describes CAM assay as an outstanding model to assess angiogenic performance of materials and drugs 47, and Mangir et al. (2019) and Eke et al. (2017) highlighted the use of the ex-ovo technique to improve the visual analysis, monitoring, and comparison of the materials under evaluation 35,48.
CAM assay allowed us to obtain preliminary performance of the heparin-loaded scaffolds. Chick embryo health and survival, as well as changes in the vascularization surrounding or attaching the scaffolds, provide useful information on the angiogenic activity of the scaffolds produced in this study.
Micrographs of the scaffolds were taken on the third and the sixth day after the implantation. These images were used to count the blood vessel growing towards the scaffolds, using the method described by Barnhill et al. (1983) for the calculation of the vasculogenic index (VIx). The vasculogenic index represents the number of blood vessels attached to the scaffold creating a steering wheel pattern 49. Table 3 shows a comparison of the average vasculogenic indexes for the different loading concentrations of the heparinized scaffold.
Sample
|
VIx
|
M0
|
33.5
|
M0.5
|
36.8
|
M1
|
32
|
M2
|
33
|
M5*
|
28.5
|
Table 3. Vascular index results according to loading concentration.
From the vasculogenic index it was observed that for most of the concentrations, the results were very similar regardless of the amount of heparin loaded. This indicates, firstly, that our scaffold is able to trigger an angiogenic response, this is mainly due to the presence of hydroxyapatite, since its bioactive properties provide the ability to interact with several molecules from the microvascular cells and their surface, improving and promoting their migration and attachment.
On the other hand, a slightly better performance from the scaffold loaded in the 0.5 mg/mL solution indicating that this loading concentration provided better angiogenic response. Similar results were observed from parallel project within our research group with injectable heparinized-chitosan hydrogels.
However, visually this is not completely notable, as we observed in Figure 10.
It is important to mention that for the highest heparin loading concentration, most of the embryos died, (survival rate of 20%). Many of them showed bleeding (leaky) vessels surrounding the scaffold as showed in Figure 11. Bleeding can be consireded an indicator of heparin overdose 50.
Whereas, the heparin release results for lower loading concentration provided a constant and sustained heparin release, providing heparin availability longer than the rest of the concentrations and without ‘overdosing’ the implanted area.
During angiogenesis, endothelial tip cells follow the angiogenic stimulus (mostly VEGF), while the proliferating endothelial cell follow the lead of the tip cells elongating the capillary sprout. With time, the new tubular structures will fuse to allow blood flow. According to the study by Ito & Claesson-Welsh (1999) 21, heparin contributes to present VEGF and FGF to their receptors in the cells , therefore a sustained availability provides enough time for heparin to bind with surrounding angiogenic factors, creating a gradient that will stimulate an angiogenic response (Figure 12).
3.7.1 CAM histology
The image in Figure 13 shows the result of the H&E staining for the retrieved samples of the CAM assay. In general, integration of the CAM tissue and the scaffold and the appearance of secondary vessels surrounding the CAM tissue (yellow arrows) was observed. No anomalies or appearance of inflammatory cells was detectable.
It is important to observe cellular infiltration into the scaffolds. It may also be stated that for the scaffolds with higher loading concentrations, the integration is not well defined, and the presence of blood vessels appear to be messy.