Microstructural differences of brin and self-assembling peptide hydrogels in dental pulp stem cell behavior: the effect of chlorite-oxidized oxyamylose

Tailored hydrogels mimicking the native extracellular environment could aid in overcoming the high variability in regenerative endodontics outcomes. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of brin and self-assembling peptide (SAP) hydrogels. Further, to assess the inuence of the microstructural differences between the hydrogels on the in vitro behavior of dental pulp stem cells (DPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and ber length, diameter, straightness and alignment) and by nanoindentation to measure their stiffness (elastic modulus). DPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity was determined using confocal microscopy imaging, and proliferation was determined using DNA quantication. Inclusion of COAM did not alter the microstructure of the brin hydrogels at the ber level, while affecting the SAP hydrogel microstructure (homogeneity) leading to ber aggregation. The stiffness of the SAP hydrogels was 7-fold higher than the brin hydrogels. The viability and attachment of DPSCs and DNA content was signicantly higher in brin hydrogels than in SAP hydrogels. The microstructural stability after COAM inclusion and the favorable DPSCs’ response observed in brin hydrogels suggest this system as a promising carrier for COAM and for application in endodontic regeneration.


Introduction
Oral health plays an essential role in our daily lives, contributing to good overall health and wellbeing. Yet, impaired oral conditions have a high prevalence, affecting almost half of the world population 1 . Dental pulp necrosis due to caries, trauma or developmental anomalies is standardly treated by lling the root canal space with bio-inert plastic-like materials, thus depriving the tooth from vascularization, an immune response and innervation. Immature teeth with pulp necrosis are rendered fragile even after treatment, and the roots fail to reach complete development.
Regenerative endodontics has attracted attention attempting to restore tooth vitality 2 . A clinical protocol that intends to reestablish the pulp-dentin complex has been developed, which is known under the synonyms pulp revitalization, root-canal revascularization or regenerative endodontic treatment (RET) [3][4][5] . This protocol involves the decontamination of the root canal space, followed by intentional laceration of the periapical tissues to form a blood clot matrix within the root canal space. This blood clot combined with endogenous growth factors and stem cells from the apical papillae (SCAPs), induce tooth root maturation through the thickening of the dentinal wall and apical closure [3][4][5][6][7][8] . Nevertheless, RET has been associated with highly variable outcomes [9][10][11] , and the histologic studies have shown that true pulp regeneration using the current protocol is di cult to achieve 4,11,12 .
Polymeric hydrogels are suitable candidates for tissue engineering and regenerative medicine (TERM) approaches including dental pulp regeneration 13 . The use of tailored hydrogels closely mimicking the native extracellular environment could aid in overcoming the high variability in the RET outcomes. Many recent studies have demonstrated that cell behavior is strongly in uenced by the cell microenvironment 14 , which is dictated by the hydrogels' composition and microstructure 15,16 . Polymeric hydrogels can be natural (biopolymers), synthetic or hybrids of the two 17,18 , with several advantages and disadvantages related to each class 19 . Fibrin is a typical natural hydrogel, and it has been extensively used as a biomaterial for different TERM and clinical applications 13,[18][19][20] . Fibrin is a tailorable hydrogel system utilizing brinogen, thrombin and Factor XIIIa. Fibrinogen, a soluble 340-kDa clotting factor, is enzymatically converted, in the presence of Ca 2+ , to brin monomers by the protease thrombin 21 . These brin monomers will undergo self-assembly and lateral aggregation to form proto brils that are packed into bers forming branched brous networks 21 . Factor XIIIa promotes the formation of covalent bonds between brinogen peptides to form a mesh network of brin bers 18 . The brous network and mechanical properties of brin can be tuned by altering the composition 22 . For instance, higher concentrations of factor XIIIa result in increasing the stiffness of brin by catalyzing brin covalent crosslinking and compacting bers 23 . Moreover, ber diameter and length are inversely proportional to thrombin concentration 18 , whereas increasing factor XIIIa concentrations lead to increased packing of proto brils within the bers 23 . Self-assembling peptide (SAP) hydrogels belong to the synthetic class and are produced using amino acids 24 . These peptides self-assemble to form nano brous hydrogels in physiological conditions. This self-assembly depends on the speci c amino acid sequence of the peptide.
These scaffolds consist of >99% water, with bers thought to be around 10 nm in diameter and 5− 200 nm pores, closely mimicking the natural extracellular matrix (ECM) 24 . Arginine-alanine-aspartic acidalanine-16 (RADA-16) is a member of the self-assembling peptide family, consisting of 16 residues, and can undergo self-assembly to form nano bers by forming stable β-sheet structures within physiological saline, which in turn form an interwoven nano brous hydrogel 25 . The SAP (RADA-16) hydrogel has been used in several dental pulp tissue engineering studies with variable degrees of success 13,26,27 .
This study aimed to evaluate the effect of the inclusion of a macromolecule, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of tailored brin and SAP hydrogels. COAM is a polyanionic polysaccharide derivative that acts as an antibacterial 28 and antiviral agent 29,30 and as an immunomodulator by interference with glycosaminoglycan (GAG) binding of chemokines 31 . Further goals were to assess the in uence of the microstructural differences between the hydrogels on the in vitro behavior of dental pulp stem cells (DPSCs) and to identify the most suitable hydrogel for further in vivo experiments.

Results
COAM did not modify brin microstructure, but affected SAP hydrogels leading to ber aggregation Inclusion of COAM did not alter the microstructure of the brin hydrogel at the ber level as demonstrated by AFM images ( Figure 1A&B) (Figure 2A-D) and quantitative analysis (Table 1). SAP hydrogel microstructure (homogeneity) at the ber level was affected by the inclusion of COAM ( Figure 1C&D) ( Figure 2E-H), leading to ber aggregation. SEM images further con rmed the microstructural stability of the brin hydrogels ( Figure 1E&F), whereas the effect of COAM inclusion on the morphology of the SAP hydrogels was not detected by SEM ( Figure 1G&H). The ber height distribution showed no signi cant effect of COAM inclusion on brin hydrogels ( Figure 2A&B) without COAM and ( Figure 2C&D) with COAM. SAP hydrogels without COAM ( Figure 2E&F) showed a distinct ber height distribution with three peaks between 1 and 5 nm; after COAM inclusion ( Figure 2G&H) those three peaks disappeared con rming aggregation at the ber level.
Topographic and quantitative microstructural analysis for the AFM images showed that both brin and SAP hydrogels have a nano-brous structure at different scales ( Table 1). The roughness average (Ra), was 8.1 (SD: 1.6) nm for brin hydrogels and 1.2 (SD: 0.1) nm for the SAP hydrogels. The inclusion of COAM in brin increased the Ra to 16.9 (SD: 7.5) nm. However, this increase was not statistically signi cant (p > 0.05). The mean ber diameter for the brin without COAM was 146.6 ± 1.1 nm and 156.6 ± 1.2 nm with COAM (p > 0.05). The mean ber diameter for the SAP hydrogels 73.2 ± 0.3 nm. For the SAP COAM hydrogels the ber measurements were unreliable due to ber aggregation ( Figure 1D); therefore, this was not reported.

Effect of COAM inclusion on brin and SAP hydrogel stiffness
The elastic modulus for brin hydrogels at 3.5 mg/ml brinogen concentration was 752 ± 13 Pa before and 683 ± 6 Pa after the inclusion of COAM ( Figure 3). Furthermore, for the SAP hydrogels at 3.5 mg/ml RADA-16 concentration, the elastic modulus was 5425 ± 295 Pa before and 4821 ± 386 Pa after the inclusion of COAM ( Figure 3). The stiffness of the SAP hydrogels was 7-fold higher than the brin hydrogels (p < 0.05). Inclusion of COAM did not alter the stiffness of the brin and SAP hydrogels (p > 0.05).

DPSCs show higher viability and better attachment in brin hydrogels
The viability of the DPSCs in maintenance medium was signi cantly higher in brin hydrogels compared to in SAP hydrogels at each time point (p < 0.05) ( Figure 4E). The viability did not signi cantly decrease over time for either the brin control hydrogels or brin with COAM hydrogels (p > 0.05). The average DPSC viability in the brin hydrogels was 91.3% ± 0.004 without and 89.9% ± 0.009 with COAM over the 7-day test period. On the contrary, the viability declined signi cantly in the SAP hydrogels without COAM from day 1 (66.9% ± 0.03) until day 7 (54.1% ± 0.02) and with COAM from day 1 (68.3% ± 0.05) until day 7 (53.9% ± 0.01) (p < 0.05) ( Figure 4E).
In addition, confocal images ( Figure 4A-B) and circularity evaluation ( Figure 4F) showed that DPSCs adopt a spread morphology in the brin hydrogels both with and without COAM. The average circularity score in the brin hydrogels without COAM was 0.25 ± 0.05, and 0.26 ± 0.02 with COAM, over the 7-day test period. In contrast, DPSCs remained rounded in the SAP hydrogels, again in both with and without COAM ( Figure 4C-D). The average circularity score in the SAP hydrogels without COAM was 0.76 ± 0.01, and 0.74 ± 0.02 with COAM, over the 7-day test period. Only hydrogel type had an in uence on cellular morphology (p < 0.05) indicating superior cell attachment in the brin hydrogels ( Figure 4F). DPSCs morphology did not change signi cantly with time for either brin or SAP hydrogels with and without COAM ( Figure 4F).

DNA quanti cation
The DNA quanti cation of the different cell-laden hydrogels showed signi cantly higher DNA content in brin hydrogels compared to the SAP hydrogels (p < 0.05) ( Figure 5). The ANOVA model showed a statistically signi cant relationship between the hydrogel type and the presence of COAM and the amount of DNA quanti ed (p < 0.05). DPSCs showed a proliferative pattern in brin hydrogels with an average 1.3-fold increase in DNA content at day 7 compared to day 0 for brin without COAM (p > 0.05) and a signi cant 2.1-fold increase for brin with COAM (p < 0.05).
A low DNA content was observed in SAP hydrogels without COAM at day 0 with 10-fold and 8-fold lower DNA content compared to the brin hydrogels without COAM (p < 0.05) and SAP hydrogels with COAM (p > 0.05). SAP hydrogels with COAM showed higher DNA content than SAP hydrogels without COAM ranging between 5-fold higher at day 1 and 3-fold higher at day 7. However, these differences were not statistically signi cant (p > 0.05). Moreover, the DNA content was stable in both SAP hydrogels from day 1 up to day 7 ( Figure 5).

Discussion
This study characterized the structural and mechanical characteristics of selected compositions of brin and SAP hydrogels. Nano-to micro-scale structural and mechanical cues are associated with biological responses, in both native ECM and synthetic constructs 14 . Although the understanding of the association between the surface topography and the cellular response is still limited, it has been suggested that the nano-topography enhances cellular communication in neural cell networks 32 and protein adsorption affecting the modulation of cellular interactions 33 . In this study, the nano-scale topographical features of brin and SAP hydrogels were different. The roughness average of the brin surface was 7-fold higher than the SAP hydrogel and with a 2-fold increase in the average ber diameter. Other features, such as ber straightness and alignment, were comparable. Fibrin hydrogels showed structural stability after the inclusion of our experimental macromolecule, COAM, while SAP hydrogels were affected leading to ber aggregation. The effect of COAM inclusion on the SAP hydrogels was not observed in the SEM images, which could be due to the sample preparation procedure that results in drying artifacts.
The measured stiffness of brin hydrogels, with the composition tested in the current study, was in the range of the previously reported values for the stiffness of the native pulp tissue, which has been reported to be 800 Pa 34 . In addition, the stiffness of SAP hydrogels was found to be 7-fold higher than the brin hydrogels. DPSCs are mesenchymal stem/stromal cells (MSCs) that pose the potential to differentiate into numerous cell types in vitro including odontoblasts/osteoblasts, chondroblasts, adipocytes and neuronal-like cells [35][36][37] . MSCs have been shown to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity 38 , as soft matrices induced neurogenic differentiation and stiffer matrices were osteogenic 38 . The current results might aid in explaining the observations of Hilkens et al. 26 , who reported mineralized tissue formation within SAP (RADA-16) hydrogels encapsulating DPSCs when implanted in an ectopic mouse model for 12 weeks. Future studies should explore to which extent differences in matrix stiffness would affect DPSC differentiation pro les in vivo.
The current study showed higher DPSC survival in brin hydrogels compared to in SAP hydrogels. This agrees with the data reported by Galler et al. 13 , where DPSCs in brin hydrogels at 10 mg/ml showed higher viability compared to DPSCs in SAP (RADA-16) hydrogels when evaluated using an MTT assay.
Moreover, Dissanayaka et al. 39 reported DPSCs' survival at day 4 of just above 60% in SAP (RADA-16) hydrogels at 1.5 mg/ml, re ecting the data reported in the current study. The higher cell survival in brin hydrogels can be likely explained by the presence of natural cell adhesion motives 40 facilitating cell attachment and elongated cellular morphology, which was demonstrated in the current study, while SAP hydrogels lack these cell adhesion motifs. Future studies could explore improving cellular attachment to SAP hydrogels by conjugating bioactive short peptide motives such as the integrin-binding arginineglycine-aspartic acid (RGD) to the C-terminus of the RADA-16 peptide. Another possible explanation could be the initial acidic pH (3.0) of the SAP hydrogels that is only neutralized after addition of medium to induce gelation. Differences in cell viabilities were con rmed by the DNA quanti cation results, with a 10fold lower DNA content measured at day 0 for SAP hydrogels compared to brin hydrogels. The DNA content in SAP hydrogels with COAM was 8-fold and 5-fold higher compared to SAP hydrogels without COAM at day 0 and day 1, respectively, suggesting an initial protective in uence for COAM that needs to be further investigated. DPSCs' viability and the DNA content for the SAP hydrogels with and without COAM was then relatively stable over the remaining period of the experiments, which strengthens the hypothesis that this drop in viability is related to the low attachment and the pH conditions at the time of encapsulation.
One interesting outcome was the effect of COAM on the increase of the DNA content. Such effect for the presence of COAM may be explained in terms of a biological in uence since no effect was observed for COAM inclusion on the structural and mechanical properties of the brin hydrogels. COAM is a polyanionic polysaccharide derivative with an antibacterial 28 and a broad-spectrum antiviral activity that acts as an immunomodulator 29,30 . COAM induces and binds chemokines such as granulocyte chemotactic protein-2 (GCP-2) leading to signi cant recruitment of myeloid cells in mice 41 . Furthermore, it has been demonstrated that COAM competes with glycosaminoglycans (GAGs) for binding and recruitment of chemokines 31 . This COAM-chemokine binding complex in uenced chemokine localization and selectivity of leukocyte responses and migration 31 . DPSCs and MSCs produce a plethora of soluble factors, cytokines and chemokines in uencing cellular growth, proliferation, migration, differentiation and immune responses 42,43 . For example, insulin-like growth factor-1 (IGF-1), a cytokine produced by DPSCs, was found to stimulate DPSCs' proliferation in serum-free culture medium 44 . Moreover, DPSCs overexpressing the chemokine stromal-derived factor-1 alpha (SDF-1a/CXCL12) showed higher cell proliferation compared to wild-type DPSCs 45 . Therefore, one possible explanation for the higher DNA content in brin hydrogels with COAM could be the formation of a binding complex increasing the availability of factors and chemokines involved in cellular proliferation inside the 3D hydrogel microenvironment, which is in line with preliminary experiments, in which we found that COAM binds SDF-1a/CXCL12, both on solid phase and in solution (unpublished data).
Finally, the current study presents a comprehensive structural and mechanical characterization for two promising biomaterials for dental pulp tissue engineering in combination with an analysis of biological features such viability, cell shape and proliferation. Future research will explore the in uence of different hydrogel properties such as matrix stiffness on DPSC migration and differentiation. Furthermore, the molecular mechanisms underlying the effect of COAM on DPSCs' proliferation need to be investigated in detail in order to obtain insights to optimize their use in tissue engineering.

Conclusion
The microstructural stability after the inclusion of COAM, the preservation of cell viability, elongated morphology and higher DNA content observed in the brin hydrogels suggests this system as a promising carrier for COAM and for application in endodontic regeneration.

Materials And Methods
All methods were performed in accordance with the relevant guidelines and regulations.

Structural and Mechanical Characterization
Hydrogel composition and preparation Fibrin hydrogels were prepared by mixing brinogen and thrombin components in equal volumes (pH = 6.6), as described previously 18 . Plasminogen-depleted brinogen (Enzyme Research Laboratories, USA), derived from human plasma, was dissolved in 20 mM HEPES and 150 mM NaCl ( brinogen buffer). Sterile stock solutions of thrombin (Sigma, USA), derived from human plasma, and factor XIII (Fibrogammin, CSL Behring, Germany) were prepared in 20 mM HEPES, 150 mM NaCl, 40 mM CaCl 2 and 0.1% BSA (thrombin buffer). Thrombin and factor XIII were mixed with the thrombin buffer and were kept in a water bath at 37 °C for 30 min in order to activate factor XIII to factor XIIIa. The control brin hydrogels were prepared at 3.5 mg/ml brinogen, 0.1 U/ml thrombin and 0.1 U/ml factor XIII, whereas the test brin hydrogels were prepared at 3.5 mg/ml brinogen, 0.1 U/ml thrombin, 0.1 U/ml factor XIII and 1 mg/ml COAM. SAP RADA-16 hydrogels were prepared according to the manufacturer's instructions by mixing the peptide solution (PuraMatrixÔ Peptide Hydrogel; BD Biosciences, USA) with 20% sucrose solution followed by adding an equal amount of phosphate buffered saline (PBS) for gelation. The control SAP hydrogels were prepared at 3.5 mg/ml RADA-16 peptide, whereas the test SAP hydrogels were prepared at 3.5 mg/ml RADA-16 peptide and 1 mg/ml COAM. COAM was synthesized by a two-step oxidation of amylose, puri ed and fractionated according to molecular weight (MW) as described previously 29,46 . COAM was endotoxin-free and used as MW mixture (corresponding to protein molecular equivalent weights exceeding 100 kDa).
Atomic force microscopy (AFM) AFM imaging was performed to characterize the microstructure of the different hydrogels at the ber level. A 100 ml sample from each hydrogel composition (n=3) was deposited on a silica sample holder and incubated at 37 °C for 30 min. After gelation, the top surface of the hydrogel was carefully removed using gentle air blowing/drying to allow the imaging of the inner network. Agilent 5500 with MAC III controller and JPK Nanowizard 3 AFM systems were used for morphological imaging in intermittent contact mode in air. A sharp microlever probe MSNL-F (f = 120 kHz, k = 0.6 N/m, tip radius of curvature < 12 nm) was used. The AFM topography images were leveled, line-corrected and measured (height and roughness pro les) using Gwyddion 47 . A ber extraction algorithm, ct-FIRE 48 , was applied to the AFM images to characterize the ber diameter, length, straightness and alignment.

Scanning electron microscopy (SEM)
A 100 ml sample from each hydrogel composition (n=3) was prepared then xed using 4% glutaraldehyde in PBS for 30 min. This was followed by drying in an ethanol series for the brin hydrogels and freeze-drying for the SAP hydrogels because the SAP hydrogels disintegrated in ethanol. Subsequently, the samples were attached to aluminum stubs and sputter coated with a 5 nm thick platinum layer under vacuum. The microstructure was then observed using a XL30 FEG scanning electron microscope (Philips, Panama).

Evaluation of hydrogel stiffness
The stiffness of hydrogels of each composition (n=3) was determined using a Chiaro Nanoindenter (Optics11, the Netherlands) by applying serial indentations with a spherical glass probe (r = 24.5 µm) attached to exible cantilever (k = 0.063 N/m). Loading and unloading velocities of the probe were set to 2 µm/s, with 2 s of holding period in between. For each individual sample, matrix scans (5 × 5 points) from three random locations were obtained. Load vs. displacement curves were extracted individually for each indentation point, and the Elastic Modulus (E) was calculated by using a Hertzian Contact Model (Poisson's ratio = 0.5) with Piuma Dataviewer Software (Optics11, the Netherlands), using equation (1): where F is the applied force, E is the elastic modulus, R is the radius of the probe, h is the indentation depth, and ϑ is Poisson's ratio.

Biological Characterization
Primary cell cultures Dental pulp tissues were acquired with informed consent from patients (15-20 years of age, male and female) undergoing extraction of third molars for therapeutic or orthodontic reasons as described previously 49 . Written informed consent was obtained from the patients or their parents, as approved by the medical ethical committee of Hasselt University, Belgium (protocol 13/0104U). The dental pulp tissue was harvested with forceps after mechanically fracturing the disinfected tooth with surgical chisels. Pulp tissues were then rinsed and transported at 37 °C in Eagle's Minimal Essential Medium, alpha modi cation (αMEM, Sigma-Aldrich, USA) supplemented with 2 mM L-glutamine (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich) and 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scienti c, USA). DPSCs were isolated according to the explant method and expanded in culture as described previously 49 . Cells were cultured in α-MEM, enriched with 10% heatinactivated foetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM l-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich). Only mycoplasma negative cells, screened with the PlasmoTest TM kit (InvivoGen), were used . All DPSC cultures were tested for the expression of the following (stem) cell markers at the protein level by means of ow cytometry as described previously 49 : positive for CD29,CD73, CD90 and CD105 and negative for CD31, CD34 and CD45.

Evaluation of DPSC viability
To obtain enhanced uorescent protein (eGFP) labelled cells, pooled DPSCs from three donors were transduced with a lentiviral vector encoding eGFP and a blasticidin resistance cassette. Selection was performed with blasticidin (10 µg/mL, InvivoGen, Toulouse, France). Stem cells were used until passage 15. These labelled DPSCs at 1x10 6 cells/ml seeding density were encapsulated in 100 ml hydrogels (n=9) with and without COAM and deposited in a glass bottom 96 well plate (CELLview slide, Greiner, Austria). After gelation, an equal amount of maintenance culture medium was added (αMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1% FBS). Tranexamic acid at 0.5 mg/ml (Exacyl, Eumedica, Belgium) was added to the medium of brin hydrogels to prevent brin degradation. After 1, 4 and 7 days in culture, the nucleus of the cells was labelled with Hoechst 33342 (Invitrogen, USA), and the dead DPSCs were labelled using the nucleic acid dye propidium iodide (PI) (Invitrogen) according to the manufacturer's instructions using an incubation for 15 min at 37 °C. The images were collected using laser scanning confocal microscopy (LSM 880, Zeiss, Germany) using a 20× objective (EC Plan-Neo uar 20×/0.50 M27). The uorescence excitation/emission was measured at 490/552, 597/695 and 410/490 nm for GFP, PI and Hoechst 33342, respectively. The number of live cells and dead cells were analyzed from 5 different regions per well (425 mm × 425 mm × 10 mm) in Fiji (Image J, National Institutes of Health, USA) 50 . Viability was calculated as a percent of live cells among the total number of live and dead cells.
Evaluation of DPSC circularity (shape analysis) Live cells from 5 different regions per well (425 mm × 425 mm × 10 mm) were segmented using a combination of watershed segmentation, thresholding and manual contour correction for cell boundaries. Shape (circularity) of segmented cells per region, excluding cells on the image edges, was analyzed using the particle analysis plug-in in Fiji (Image J, National Institutes of Health, USA) 50 . The circularity score was averaged for each well yielding a nal circularity score ranging between 0 and 1, where the closer the score to 1, the closer the shape to a circle, which would indicate poor cellular attachment.

Evaluation of DPSC proliferation (PicoGreen/Quant-iT DNA Quanti cation)
DPSCs at 5x10 5 cells/ml seeding density were encapsulated in 100 ml hydrogels (n= 3 per gel and per time point) with and without COAM and deposited in a 96 well plate coated with 50 ml of the same hydrogel devoid of cells or COAM (TPP tissue culture plates, Sigma-Aldrich, USA). Hydrogels devoid of cells as blank replicates were prepared. In addition, 2D controls of 5x10 3 cells were seeded in a 96 well plate. After gelation an equal amount of serum free mesenchymal stem cell (MSC) medium (MesenCultÔ-ACF plus medium, Stem Cell Technologies, Canada) supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin was added to each well. At baseline (day 0) and after 1, 2, 3 and 7 days in culture, a PicoGreen/Quant-iT kit (Invitrogen) was used to investigate the effect of different hydrogels on cellular proliferation. The DNA content of three hydrogels per condition and per time point was calculated for three independent experiments. Fibrin hydrogels were rst digested in a buffer composed of 50 FU/ml nattokinase in 5mM EDTA in PBS for 2 h at 37 °C. SAP hydrogels were digested in a buffer composed of 1 mg/ml Pronase (Thermo Fisher Scienti c, USA) in PBS for 2 h at 37 °C. The cells seeded in the control wells were released using Trypsin-EDTA. The contents of the wells were collected, and cell pellets were retrieved by centrifugation. Retrieved cell pellets were then lysed to extract DNA using 100 ml cell lysis buffer composed of 0.029% Sodium EDTA, 0.112% Sodium pyrophosphate decahydrate, 0.88% Sodium chloride, 0.315% Tris HCl, 1% Triton-X-100, 0.038% EGTA, 0.0001% Leupeptin, 0.019% Sodium orthovanadate, 0.0216% β-glycerophosphate and 1mM PMSF (ab152163, Abcam) and centrifuged at 14000 rpm at 4°C to collect the supernatant. A 200 ml working solution representing each well (hydrogel) was prepared and aliquoted directly into black 96-well plates (Chimney well, FluotracÔ, Greiner, Austria), according to the manufacturer's instructions, and incubated for 5 min protected from light at room temperature. The uorescence excitation/emission was measured at 481/520 nm using a microplate reader (VarioskanÔ, Thermo Fisher Scienti c, USA). A standard curve was performed with lDNA, provided with the kit and treated equally to the sample plates. The standards ranged from 10 ng/ml to 1 mg/ml lDNA and were used to calculate the nal DNA content per ml of sample.

Statistical analysis
Statistical analysis was performed using the statistical software package GraphPad Prism 8.00 (GraphPad Software, La Jolla California USA). Comparison of the ber measurements from AFM images was performed using a one-way analysis of variance (ANOVA). Comparison of the stiffness of the hydrogels was performed using a two-way ANOVA. The in uence of the different experimental conditions and the time factor on cell viability, shape and DNA quantity was modeled using a three-way ANOVA. All ANOVA tests were followed by Tukey's correction for multiple comparisons. Statistical signi cance was determined at p<0.05. Descriptive statistics are represented as mean and standard deviation (SD), or standard error of mean (±), where appropriate.