3.10 Mechanical Properties
The mechanical strength of the collagen based scaffold was assessed using a tensile strength test and the results are shown in Fig. 6b. As can be seen in the figure, all collagen-based scaffolds exhibited a typical stress-strain curve. The pure collagen scaffold exhibited higher elongation than other scaffolds, which implies that the collagen scaffold possesses higher flexibility than other scaffolds. In the case of metal oxide nanoparticle mediated collagen scaffold a distinctive yield point is observed when compared to collagen, EDC-NHS, and TES-PAMAM -G3 cross-linked collagen scaffold indicating a transition from an elastomeric to a tough plastic nature. The linear component of the stress-strain curves was used to measure the Young’s modulus. The tensile strength, percentage of strain, and Young’s modulus of scaffolds are presented in Table 4, Supplementary file. As seen in the table, the tensile strength, Young’s modulus and strain (%) were 0.76 ± 0.4 MPa, 7.71 ± 0.3 MPa and 8.36% respectively for collagen in comparison to the EDC-NHS- collagen scaffold which had tensile strength of 2.21 ± 0.1 MPa, Young’s modulus of 90±0.8 MPa and strain of 1.88% respectively. The TES-PAMAM- G3collagen scaffold showed tensile strength 10.93 ± 0.7 MPa, Young’s modulus of 181 ± 0.5 MPa and strain of 6.67%. In the case of TES-PAMAM- G3 functionalized ZnO, TiO2, Fe3O4, CeO2 and SiO2 nanoparticle- collagen the tensile strength was 11.56 ± 0.9 MPa, 5.54 ± 0.5 MPa, 5.68 ± 0.2 MPa, 2.84 ± 0.9 MPa, 5.51 ± 0.6 MPa and Young’s modulus of 273.14 ± 0.2 MPa, 135.30 ± 1.0 MPa, 100.01 ± 0.3 MPa, 76.49 ± 0.5 MPa, 236 ± 0.2 MPa and strain of 5.36%, 4.58%, 5.31%, 3.65%, 1.69% respectively. The Young’s modulus of MOs-TES-PAMAM- G3 NPs cross-linked collagen scaffold followed the order: ZnO > SiO2 > TiO2 > Fe3O4 > CeO2. This result suggested ZnO- TES-PAMAM-G3 mediated collagen scaffold exhibit 35 fold higher Young’s modulus than pure collagen scaffold. Compared to CeO2- TES-PAMAM-G3 mediated collagen scaffold, ZnO- TES-PAMAM-G3 mediated collagen scaffold showed 3.5 fold higher Young’s modulus (p ≤ 0.05). In addition to this, ZnO nanoparticle mediated collagen scaffold indicate higher elastic nature than other metal oxide mediated scaffolds due to their large pore size structure. The obtained Young’s modulus value of collagen scaffold in the presence of TES- PAMAM-G3-ZnO (273.14 ± 0.2 MPa) is higher than hydroxyapatite incorporated collagen scaffold (230 ± 30 MPa). It has been observed that TES-PAMAM-G3 functionalized ZnO nanoparticle mediated collagen scaffold has 92 MPa higher Young’s modulus than previously fabricated ZnO- TES-PAMAM-G1-collagen scaffold. These results suggest that TES-PAMAM-G3 functionalized metal oxide nanoparticles improve the mechanical strength of collagen scaffold due to their smaller size, large surface area leading to increase the cross-linking density on nanoparticle surface. The above results clearly suggested that cross-linking of MO-TES-PAMAM-G3 in collagen can effectively enhance the mechanical properties of collagen scaffolds.
3.11 Biodegradation of MO-TES-PAMAM- G 3 -Collagen Scaffold
The in vitro biodegradation profile of dendrimer cross-linked collagen scaffold as a function of degradation time is shown in Fig. 6c. The obtained results showed that all collagen-based scaffolds prepared in this work had higher biodegradation. However, in contrast to the collagen scaffold (98% after 30th day), the rate of weight loss was lower in the case of nanoparticle-mediated collagen scaffolds (82–90 % ± 0.32–2.5), indicating that the degradation trend is affected by the degree of cross-linking and the existence of the cross-linker. The cross-linked scaffold initially had low weight loss (up to one week) and thereafter the scaffold underwent steady degradation indicated by a decrease in weight with time. As compared to the collagen scaffold, the metal oxide nanoparticle mediated collagen scaffold has a higher degree of cross-linking and is more resistance to degradation in PBS medium at 37oC. The formation of covalent bonds between collagen and MO-TES-PAMAM-G3 NPs may be possible reason for higher scaffold strength and for a longer period of time.
3.12 Swelling Degree of MO-TES-PAMAM-G 3 -Collagen Scaffold
The swelling degree or water uptake is a significant parameter that represents the efficiency of oxygen and nutrient transfer inside the scaffold. Swelling degrees of collagen, EDC-NHS-collagen, TES-PAMAM-G3-collagen, and MO-TES-PAMAM-G3-collagen scaffolds in water medium are showed in Fig. 6d. Generally, highly porous collagen scaffolds have a very high swelling degree, compared to less porous scaffold. As shown in the figure, collagen, TES-PAMAM-G3-collagen, and MO -TES-PAMAM-G3 - collagen scaffolds possess higher swelling capability. However, the swelling degree of the collagen scaffold was much higher than the metal oxide nanoparticle mediated collagen scaffold because of the hydrophilic properties of the carboxylic group and its porous structure, which can greatly influence the scaffold swelling degree. Among the different metal oxide nanoparticle mediated collagen scaffolds, there is no significant difference in swelling behaviour. This finding was corroborated with reports of Ullah et al. have reported that the swelling ratio of collagen was decreased in the presence of ZnO nanoparticle.This effect may be explained by the metal oxide nanoparticle interacting with the collagen fibre and serving as a filler to fill the gap space inside the scaffold network, resulting in a more compact scaffold that does not swell as much as a pure collagen scaffold.
3.13 Quantification of Cross-linking degree between collagen and MO-TES-PAMAM–G 3 NPs
The degree of cross-linking in collagen-nanoparticle interaction was quantified from a number of activated carboxylic acid groups in EDC-NHS reaction, and obtained results are shown in S5, Supplementary file. The collagen sample indicated 96 ± 2.88% of activated carboxylic group, inferring that EDC reagent not activate 100% of the carboxylic group in collagen. The EDC cross-linked collagen sample showed that 23±2.19% of the activated carboxylic group was consumed by the cross-linking reaction. In the case of TES-PAMAM-G3 and MO- TES-PAMAM –G3 cross-linked collagen 66 ± 0.90%, 84 ± 0.36%, 82 ± 0.42%, 84 ± 0.36%, 81 ± 0.45% and 83 ± 0.39% of activated carboxylic acid group was consumed by cross-linking reaction for ZnO, TiO2, Fe3O4, CeO2 and SiO2 nanoparticle respectively. This result reveals that a large number of the activated carboxylic group was consumed when MO -TES-PAMAM-G3 were introduced to the EDC-NHS cross linking reaction, resulting in a higher degree of cross-linking through the carboxylic acid groups of collagen with free amine groups of dendrimer, thus leading to the improved extent of reaction.
To investigate the effect of concentration of MO-TES-PAMAM-G3 nanoparticle in collagen scaffold on the biological behaviour of cells, Keratinocyte was employed to assess cell viability. The cell-viability of collagen, EDC-NHS- collagen, TES-PAMAM-G3 -collagen, and MO -TES-PAMAM-G3-collagen-based scaffolds were analyzed through MTT assay, and observed results are showed in S6, Supplementary file. As can be seen in the figure, the cell viability of collagen scaffolds was highly depended on the concentration of TES-PAMAM-G3 and MO-TES-PAMAM-G3. The cell viability of TES-PAMAM-G3 cross-linked collagen scaffold decreased gradually as the concentration of TES-PAMAM-G3 increased, which was more pronounced at higher dendrimer concentrations. At higher concentrations, the cationic TES-PAMAM-G3 dendrimer may lead to cellular bonding through electrostatic attraction with negatively charged cells. Due to the high concentration of dendrimer, excessive cell bonding or severe cell membrane damage can occur, resulting in the low cell viability observed. However, up to 50 µM concentration of dendrimer, cells are viable in a collagen scaffold. This observation was consistent with other reports, where in 10–100 µM PAMAM induced minimal cytotoxicity and use of 50 µM of PAMAM for the cross-linking of collagen scaffold was appropriate, especially when the release of dendrimer from the scaffold was relatively slow during the biodegradation of collagen. Up to 100 µM, the metal oxides nanoparticle cross-linked collagen scaffold showed 80% of cell viability. This observation implies that after the functionalization of TES-PAMAM-G3 on the nanoparticle surface, the cell viability was enhanced when compared to dendrimer alone.
3.15 Wound healing study of MO-TES-PAMAM-G 3 - Collagen Scaffolds
The wound healing efficiency of the collagen-based scaffold was investigated through in vivo animal test on Wistar Albino rats. For this collagen, TES-PAMAM-G3- collagen, and MO- TES-PAMAM-G3- collagen scaffolds were implanted on the wounded area and wound healing efficiency monitored periodically (on days 7, 14, 21, 28) and percentage of wound closure was measured (Fig. 7a-b). For the comparison of wound healing process, saline water treated open wound was kept as a control. As seen in the figure, the groups that were treated with metal oxide nanoparticles had stronger wound healing properties than the other groups all of the time.
On day 7, there was 62–79% wound contraction in the metal oxide nanoparticle treated groups, whereas in control, collagen, TES-PAMAM-G3- collagen scaffold treated groups wound contraction was observed to be 28%, 46% and 54% respectively. On day 9, wound contraction (90%) was observed in ZnO mediated collagen scaffold compared to other metal oxide nanoparticles. The wound contraction was enhanced in all the groups with no sign of inflammation on day 14. In presence of metal oxide nanoparticle treated groups, the wound contraction nearly reached 71–96%, while the wounds of the control and other groups were not healing well. On day 21 and 28, the same tendency was observed with metal oxide nanoparticle treated groups. The increase in wound healing efficacy observed with metal oxide nanoparticle treated scaffolds due to their smaller size with better antibacterial activity through electrostatic interaction of positively charged metal oxide with negatively charged bacterial membrane leads to release the reactive oxygen species (ROS) on surfaces of the nanoparticles, which result in damage bacterial cell (Fig. S7-S10, Supplementary file). Among the different metal oxide nanoparticle treated groups, ZnO exhibited higher wound contraction followed by TiO2 and CeO2 nanoparticle. Although, the difference in percentage of wound contraction between Fe3O4 and SiO2 nanoparticles were negligible (p ≥ 0.002). The wound healing efficiency of metal oxide nanoparticle followed order: ZnO > TiO2 > CeO2 > Fe3O4 > SiO2 NPs.14 nm size of ZnO-TES-PAMAM-G3 mediated collagen scaffold showed large wound contraction (90%) on day 9, whereas previously fabricated 40–75 nm size of ZnO-TES-PAMAM-G1 mediated collagen scaffold (84%) on day 14. This accelerated wound contraction can be attributed to 14 nm size of ZnO –TES-PAMAM-G3 nanoparticle coupled with larger surface areas, which result in higher cross –linking density with collagen. This cross –linked collagen scaffold exhibited higher mechanical strength, pore size, better cell – viability and antibacterial activity leads to acceleatate wound healing process much faster than other metal oxide nanoparticle.
3.16 Histopathology and Masson’s trichrome stain study
Furthermore, both histopathological and masson trichrome-stained wound sections were examined under the microscope to confirm the quality and maturity of the healing nature of tissue on different days. Figure 8a (10x magnification) shows H & E stained sections of healing wounds from control, collagen, TES-PAMAM- G3 -collagen, MO -TES-PAMAM -G3 - collagen treated groups on days 7, 14 and 21 post-wounding. On day 7, the wound treated with TES-PAMAM-G3 -collagen, and MO -TES-PAMAM- G3-collagen scaffolds contained fewer inflammatory cells when compared to the collagen scaffold treated group and control. In addition to this, on day 7 sparsely grown blood vessels were formed perpendicular to the wounded area in all groups. In the case of control and collagen scaffold treated group, no re-epithelialization occurred whereas TES-PAMAM-G3 -collagen and MO -TES-PAMAM- G3- collagen treated groups showed re-epithelialization layer in wounded area and exhibited lesser wound contraction. After 14 days, a continuous nascent epithelial layer was formed in all the groups, namely neo-epidermis. Although the nanoparticle-mediated collagen scaffold treated wound parts still had a few inflammatory cells, they also showed fibroblast and blood vessel formation, as well as collagen synthesis. As can be seen in the figure on day 14, the wound contraction was greatly improved in all groups, in particular, ZnO nanoparticle treated group showed higher wound contraction than other groups.
On day 21st, a continuous nascent epithelial layer and some collagen fibers began to grow over the nanoparticle-mediated collagen scaffolds-treated wounds, indicating complete healing. Furthermore, histopathological images of nanoparticle-mediated collagen scaffold revealed more matured dermis and epidermis layers when compared to control groups. The non-treated group had a slower epithelialization rate and less collagen bundle development, as well as irregular collagen fibre packing. The average gap length between newly formed epithelium in TES-PAMAM- G3- collagen scaffolds treated group was lower than collagen and control groups. On day 21, metal oxide nanoparticle treated groups showed complete wound contraction than other scaffolds, especially ZnO nanoparticle treated group exhibited completely closed wound. The enhanced wound healing behavior of nanoparticle –mediated collagen scaffold treated groups may be attributed to a combination of factors including a favourable bioactive environment for re-epithelialization, antimicrobial growth inhibition, fluid handling properties, and moisture vapour permeability of the scaffold, all of which could provide an ideal moist environment around the wound site for accelerated healing.
On the other hand, Masson’s trichrome staining of TES-PAMAM-G3 and ZnO -TES-PAMAM-G3 treated collagen scaffolds were indicated (Fig. 8b) the formation of fair and typical collagen layer with large macrophage and fibroblast density. This observation inferring that wounds treated with ZnO-TES-PAMAM-G3 NPs healed faster in comparison to all other groups due to more number of fibroblast, keratinocyte migration and collagen deposition in wound site leading to acceleration in the healing process.
3.17 Quantification of released metal ion from scaffold - ICP-OES Analysis
The metal ion content of TES- PAMAM-G3 functionalized MO nanoparticle mediated collagen scaffolds implanted skin is presented in Table 5, Supplementary file. The obtained results show that Zn, Ti, Fe, Ce, and Si ions were presented in the healed skin in very small quantity such as 0.12, 0.10, 0.91, 0.06, and 0.15 ppm. The iron oxide nanoparticle has highly leached from scaffold material when compared to other metal oxide nanoparticle. There is no significant difference noticed in terms of ion leaching between ZnO and CeO2 nanoparticle (p value 0.001). It has been reported that minimum inhibition concentration (MIC) of different metal oxide nanoparticles against S.aureus and E.coli bacteria strain exhibited 0.0039–0.0312 mg/mL, 0.0097–0.0195 mg/mL, 6.25–12.5 mg/mL, 2.15-10.0 mg/mL and 0.0006 mg/mL for ZnO, TiO2 and Fe3O4, CeO2 and SiO2 NPs respectively[51–56]. The obtained concentration of leached metal ions from collagen based scaffold showed much lower than the MIC concentration of metal oxide nanoparticle. This observation clearly implies that the antibacterial activity of metal oxide nanoparticle responsible for generation of reactive oxygen species on the nanoparticle surface. In this study, ZnO-TES-PAMAM-G3 mediated collagen scaffold exhibited lesser leaching (0.12 ppm) than previously fabricated ZnO-TES-PAMAM-G1 mediated collagen scaffold (0.37 ppm). The decreasing the ion leaching in the case of smaller size of spherical shape of ZnO nanoparticle due to their higher cross- linking density leading to stronger interaction (higher cross-linking density) with collagen fiber. The obtained decrease in the quantity of metal oxide nanoparticle in comparison to added quantity could be due to the loss of particles during scaffold preparation. Further, these small quantities of nanoparticle (size > 10 nm) can be clear out via liver and the mononuclear-phagocyte system (MPS).