*2.10. Statistical Analysis*

All experiments were repeated at least 3 times independently with 3 repetitions within each experiment. The results are presented as averages +/− standard deviations (*n* = 0–5) or errors

(*n* = 6+). Statistical significance was assessed using one-way or two-way ANOVA and Tukey's HSD post-hoc tests using RStudio (RStudio Inc., Boston, MA, USA) and plotted using the ggplot2 package. Statistical significance was considered to be achieved with *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Laser Two-Photon Polymerisation*

As mentioned in the introduction and methods, the possibility of structuring the Al-based hybrid material has already been described previously [21]. However, we wanted to further demonstrate our ability to fabricate complex 3D shapes and thus have fabricated a micro-structure reminiscent of Zerg (StarCraft, Blizzard Entertainment ©, Irvine, CA, USA)—see Figure 1. High volume suspended features were reproducibly fabricated, like the claws of the Zerg hydralisk. Literature shows that by precisely controlling the 3D cell microenvironments, one can achieve complex cellular responses, like stem cell homing towards artificially designed niches [31]. Another important group of factors in designing artificial cell niches are associated to the material chemistry and properties [32]. Integration of several materials in a single structure will hopefully one day help to guide stem cell di fferentiation towards di fferent lineages on the same sample.

**Figure 1.** Right, A 3D micro-structure reminiscent of Zerg fabricated using a commercially available Nanoscribe Photonic Professional GT system. The scale bar corresponds to 10 microns. Left, A CAD model of the structure.

Multiple materials can be structured with laser two-photon polymerisation and the Al-based hybrid organometallic polymer presented in this work expands the selection of possible cellular niche materials even further. To date, a number of reports have been made of multiple-material structure fabrication using laser two-photon polymerization—including some of our own, in which PDMS, hybrid organometallic polymer based on Zr, PEG-DA and commercially-available OrmoComp have been integrated together [12]. Lamont et al. have developed a technique to exchange the material that is being used to fabricate structures on-the-go, allowing up to five di fferent materials to be incorporated within the same structure with increased fabrication speed and potentially, multiple functionalities for applications like drug delivery, advanced optics, meta-materials and microrobotics [33]. Additionally, two or even more disparate fabrication techniques can be employed within the same structure as demonstrated with fused filament fabrication 3D-printed structure modification using laser ablation [34].

The addition of ye<sup>t</sup> another 3D-printable material with new properties to the engineer's menu will allow for a wider variety of new structures and applications to be envisaged, particularly in fields that require high precision micro-fabrication, like tissue engineering sca ffolds and drug delivery devices.

#### *3.2. Protein Adsorption Assay*

Extracellular matrix protein adhesion to the di fferent surfaces may a ffect the subsequent adhesion of cells to the surfaces. Collagen type I was chosen as a model system for this experiment—its adsorption to simple spin-coated samples was assessed due to collagen type I abundance in the extracellular matrix. Collagen supports cellular adhesion via RGD sequences, which are specifically recognised by cellular adhesion proteins integrins. These interactions facilitate strong integration between a tissue engineered sca ffold and the surrounding tissues, so it is important to have a material that supports collagen adsorption to its surface well for applications where cell adhesion is beneficial.

Collagen itself has been used as a tissue engineering building block in various studies, for example that of Ber et al., where osteoblast growth was guided on 2D surfaces of collagen [35] or work by Ramanathan et al., who have investigated 3D hybrid collagen matrixes as antibacterial dermal substitutes [36]. Collagen and its derivatives are a widely investigated group of materials for soft tissue engineering, while for hard tissue, like bone, collagen coating is a highly desirable approach that can be used in conjunction with other materials. Multiple reviews have focused on the use of collagen as a tissue engineering material either in a pure or composite form [37–39]. Consequently, a possibility of coating hybrid polymer structures with collagen seems like a good approach for improving cellular integration and therefore chosen in this study.

In our experiments, the choice of protein quantification technique was limited by residual photo-initiators within the materials which rendered them fluorescent. Thus, the signal of fluorescence-based techniques for protein visualisation would have been hindered by sample autofluorescence. To overcome this issue, a colorimetric assay was employed.

Collagen adsorption was measured on both the hybrid polymers and reference glass slides by immersion in a solution of collagen, then dying the bound collagen and measuring the optical absorption of the dye. We found that even though the largest amount of collagen was found on the Zr surface and the smallest amount on Al surface, the results in each group were highly disperse, and no statistically significant di fferences were determined with *p* = 0.35 between Al and Zr, *p* = 0.72 between Glass and Zr and *p* = 0.81 between Glass and Al. The results are presented in Figure 2.

**Figure 2.** Collagen I adsorption to Al and Zr based hybrid organometallic polymers and glass. The results are presented as average Sirius Red absorbances + standard errors for three independent experiments with n = 3 per each experiment (a total of n = 9 per material).

A calibration curve between the total amount of collagen I and Sirius Red absorption was prepared and is presented in Supplementary Materials Figure S1. A highly linear relation was found in the range of 0–100 μg with an R<sup>2</sup> value of 0.994. The amounts of collagen adsorbed to the samples were calculated according to the linear model and were found to be 5.59 μg for Glass, 4.40 μg for Al and 6.73 μg for Zr on average per sample.

In our previous work [21], we investigated the contact angles of these materials and found them to be 39◦ for Glass, 72◦ for Al-based hybrid materials and 71◦ for Zr-based hybrid materials. The collagen adsorption data does not seem to correlate with the surface contact angles. Even though there is a significant di fference between the contact angle of glass to Al or Zr surfaces, the di fference in collagen I adsorption could not be distinguished due to a high deviation from the mean. A paper by Ying et al. [40] indicates that around a two-fold increase in the adsorption of collagen I to the glass surface is expected with an increase of the surface contact angle from 40◦ to 70◦. An analogous result would be expected on other surfaces, since protein adsorption is governed by the same forces as surface contact angle—electrostatic and hydrophobic interactions.

## *3.3. Cell Viability*

Having established the structurability of the materials as well as their collagen adsorption capacity, we investigated how their surfaces influence cellular viability. In this particular study, a primary rat muscle cell line was used to model a situation that would be as close as possible to a clinical one. Using primary muscle-derived stem cells is a safe choice in designing a possible future tissue engineering strategy due to the abundance of donor tissue sites, relatively mild donor site morbidity and high proliferative capacity as well as multipotency of the cells. A grea<sup>t</sup> example by Nieponice et al. demonstrates how rat muscle-derived stem cells could be used together with elastomeric sca ffold-building materials in constructing vascular grafts [41]. The study successfully showed that the cells tended to home to the lumen of the vascular grafts and started to express α-actin, calponin as well as secrete collagen. Endothelial di fferentiation was supported via the presence of von Willebrand factor. Our isolated rat muscle-derived progenitor cells were positive for CD34, Myf5 and c-kit and negative for CD45 (Figure S2).

This experiment aimed to find the ratios between the numbers of viable, early apoptotic, necrotic and late apoptotic cells at di fferent timepoints and on di fferent surfaces. Flow cytometry data shows that no statistically significant di fferences between the numbers of cells in each category can be observed, since the majority of cells at each time point was viable (Figure 3). Additional data is presented in the Supplementary Materials, providing light microscopy images of cell culture at di fferent time points as well as statistical analysis (Figure S3 and Figure S4 in the Supplementary Materials).

The results are in accord with our previous work on these materials with NIH/3T3 cells, which showed that the cells remain viable in culture for extended periods of time [21]. Data obtained in this experiment open the way for the future applications of both Al and Zr based hybrid materials. Long-term viability support of the cells is the first step towards engineering adequate implants for patients.

#### *3.4. Cell Proliferation and Metabolism*

As a subsequent step after viability assessment, the rate of cellular metabolism was measured using MTT assay. This test is a direct indicator of mitochondrial activity, since the dye is being digested by mitochondrial reductase to yield a colorimetric signal. This can be used as an indicator for overall cellular metabolism. It is important to know if cellular metabolism rates are di fferent among the various surfaces, since significant deviations in cellular metabolism rates could potentially yield new properties of the cell population on that particular surface. Increased rates of metabolism could be associated with malignant formations and dysregulation of the cell cycle, while down-regulated rates of metabolism could indicate low cellular viability or transfer into a more quiescent state via di fferentiation or other mechanisms [42].

**Figure 3.** Flow cytometry data on cellular viability on Glass, Al and Zr surfaces after 24, 48, 72 and 96 h. The four quadrants in each case show the following: lower left—viable cells, upper left—necrotic cells, upper right—late apoptotic cells, lower right—early apoptotic cells. The lower left quadrant corresponds to 95% of all signals in all cases.

Light microscopy images show cells grown on both the hybrid polymer and glass surfaces to have formed confluent monolayers after 48 to 72 h (Supplementary Materials, Figure S3). No significant differences were observed neither in daily culture check-ups nor in the images taken of those cells. The cells had healthy spindle-shaped morphologies and stopped dividing after having reached confluency.

The assessment of total metabolic activity revealed that the metabolic rate of cells grown on glass was significantly higher than on the hybrid organometallic polymers after 96 h of culture (*p* < 0.001) and significantly higher than on the Al-based hybrid starting from 48 h (*p* < 0.05). These results are presented in Figure 4. Our previous study [21] investigated the proliferation rate of NIH/3T3 fibroblasts on analogous surfaces after 120 h and showed the Zr surface to support the highest rate, while no statistically significant differences were observed between Glass and Al.

The results obtained in this experiment show promise in using both the Al and Zr hybrid polymers in tissue engineering applications. Both materials supported cell growth, even though the total rate of metabolism was lower than on reference glass surfaces.

**Figure 4.** Cell proliferation as measured by MTT. The results are presented as average +/- standard error. A total of 9 measurements were performed per material per time point (3 independent experiments with 3 repetitions in each). #—significant difference between glass and Al with *p* < 0.05; \$\$\$—significant difference between glass and Al with *p* < 0.001; &&&—significant difference between Al and Zr with *p* < 0.001; @@@—significant difference between glass and Al with *p* < 0.001; §§§—significant difference between Al and Zr with *p* < 0.001.

#### *3.5. Cell Adhesion Strength*

Cellular viability, proliferation and adhesion are highly intertwined processes. Knowing that the viability does not change with surface and that the rate of cellular metabolism grown on the Al hybrid is lower, we sought to understand if the reduced metabolic activity was associated with weaker adhesion. The cells were seeded on the samples and then shaken vigorously at two timepoints. The number of remaining cells was counted and calculated against the number of cells on reference surfaces without shaking. The results are presented in Figure 5. There was a clear trend that showed a decrease in the number of adhered cells after 24 h, suggesting that the initial stage of attachment after 4 h was stronger. Even though there were substantial differences between the number of adhered cells after 24 h, the differences were not statistically significant. However, in spite of a lack of statistical significance, the number of cells that remained attached to the Al surface was the lowest and thus in accord with the proliferation data, that showed the lowest rate of MTT metabolism on the Al- based hybrid. Overall, the Al surface seems to be slightly less supportive of cell adhesion, though insignificantly. The reason for that could potentially be the lowest adsorption of collagen to the Al surface, as presented in Figure 2. To further investigate this difference, an experiment was devised to assess the expression of adhesion-associated proteins.

#### *3.6. FAK*/*Akt Expression and Activation*

When integrin-mediated cell adhesion takes place, a cascade of biochemical reactions is activated that delivers a signal about cellular attachment to the cell nucleus, which in turn activates the transcription and translation of certain proteins that are required for these processes. The cascade is activated when cell surface integrins dimerise after binding to a specific adhesion sequence in the ECM [43]. To initiate intracellular signalling integrin dimer induces conformational changes in focal adhesion kinase (FAK), which is one of the first proteins to join the newly forming focal adhesion. Subsequently, this leads to the recruitment of multiple other adhesion-associated proteins to the focal adhesion site. Upon formation of a focal adhesions, multiple pathways can be activated, one of which is the Akt pathway. This pathway is responsible for transferring a survival and proliferation signal to the nucleus via the mTOR pathway [44]. Akt can be activated by either phosphorylation on Ser473 or Thr308—this depends on the phosphorylating protein. In the case of integrin-mediated adhesion, PI3K is being activated and thus, only Ser473 is phosphorylated [45]. Phosphorylation on the Thr308 occurs from mTORC2, which is associated with cellular metabolism and cytoskeletal reorganisation.

To assess the expression and activation of adhesion-associated FAK pathway proteins, a Western blot analysis was performed on cells grown on the investigated Glass, Al and Zr surfaces. The results are presented in Figure 6 with membrane images presented in Supplementary Materials Figure S5. Even though no statistically significant results were observed on the investigated surfaces, a tendency for reduced phosphorylation of Akt kinase can be seen in most of the cases when comparing 4 and 24 h. This is to be expected as the adhesion-associated Akt kinase's role is mostly pronounced during the initial cell-to-surface and cell-cell interactions that take place upon cell seeding.

**Figure 5.** Evaluation of cell adhesion strength on the tested surfaces. The results are presented as a ratio between the average number of cells after shaking to that of cells before shaking after 4 and 24 h of culture + standard errors. A total of 9 measurements per material were performed (3 independent experiments with 3 repetitions each). No statistically significant differences were observed between the different time points on each surface.

**Figure 6.** Western blot data, showing the expression of FAK and the expression and activation of Akt kinase after 4 and 24 h of culture on glass, Al and Zr surfaces. The amount of p-Akt (Ser 473), p-Akt (Thr308), Akt and FAK were observed. The results are presented as relative expression +/- standard error. Three independent experiments were performed. No statistically significant differences were observed.

The ratio between FAK and Akt kinases was mostly unchanged between time points and only slightly and insignificantly lower on Zr surfaces. This is again to be expected as the total amount of FAK and Akt kinases should remain unchanged.

On the glass surface, the cells tended to attach stronger during the initial 4 h—this is in accord with a higher level of Akt kinase activation in this time point. After 24 h the drop in the number of adhered cells and p-Akt (both Ser473 and Thr308) is clearly visible, though insignificant.

On the Al surface, the situation is similar in terms of p-Akt (Thr308), where the amount of phosphorylated Akt drops after 24 h in accord with a decreased number of cells. However, this was not observed in the p-Akt (Ser473) test. The level of FAK decreased as well, suggesting that the number of focal adhesions had been reduced as well.

The highest drop in terms of level of phosphorylation was observed on the Zr surface. From 4 to 24 h, the level of p-Akt (Ser473) dropped with a *p* = 0.25 and the level of p-Akt (Thr308) dropped with a *p* = 0.67. A slight, but insignificant increase was observed in the level of total FAK. Even though the changes in phosphorylation of Akt on Zr surfaces was the highest, the change in observed cell detachment was lowest in this case.

Even though some tendencies in cell attachment and adhesion-associated protein expression can be seen, these are not statistically significant.

Strong cellular adhesion is an essential prerequisite for successful tissue engineering techniques as discussed in a review by Lee et al. [46]. Whenever adhesion-associated signalling is insu fficient, anchorage-dependent cells undergo anoikis, a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding ECM.
