Biological activity of a chitosan-carboxymethylcellulose-zinc oxide and calcium carbonate in 3D scaffolds stabilized by physical links for bone tissue engineering

Abstract

Today, regenerative osteogenesis represents a clinical need, due to the incidence of bone defects that involve groups of pathologies ranging from congenital anomalies to traumatic injuries, as well as problems presented surgically. This is why the design of a polymeric biomaterial (scaffold) of chitosan, carboxymethylcellulose, zinc oxide, and calcium carbonate with similar characteristics in terms of composition and bone structure offers high potential to help address this health problem. The technique for obtaining the scaffolds of this research was to develop a physical hydrogel to have the biofunctionality of the active groups of the polymer chains used, then make use of the lyophilization process to obtain three-dimensional (3D) porous scaffolds. The physicochemical and biological properties of the scaffolds were evaluated. The scaffolds presented morphology with pore size and interconnectivity that favor the need for cell proliferation and viability. The biocompatibility tests confirm that the designed scaffolds do not present cytotoxicity and the analyzes with alizarin red staining show calcium deposits in the materials with CaCO₃ and ZnO. Osteoinduction assays to osteogenic lineage using runt-related transcription factor type 2 (RUNX2) and collagen type 1 (COL-1) antibodies allowed expression in differentiated cells. Therefore, the calcium carbonate-containing scaffolds stabilized by physical bonds have characteristics of being non-cytotoxic, bioactive, and osteoinductive, which motivate their use in future tests to evaluate their demeanor with rat models for bone engineering studies.

Keywords

Chitosan-carboxymethylcellulose-zinc oxide-calcium carbonate 3D-scaffold human dental pulp stem cells biomaterial bone-tissue-engineering

Introduction

In recent years, important advances have been made in organ transplantation, surgical reconstruction, and the use of prostheses to help repair the loss or failure of an organ or tissue. However, some drawbacks related to these treatments have led to a greater emphasis on tissue engineering using cells and scaffold-constructs.^1,2 Currently, work is being done to design practically all-human tissues and thus meet the needs we face. Also, research is being done in the area of tissue engineering using cell-based therapies and scaffolds to repair bone, cartilage, and intervertebral discs.³ The main focus of tissue engineering is its use to repair or regenerate damaged tissue by replacing tissue designed with the objective that it contributes to the restoration of the functions of the damaged tissue during regeneration and subsequent integration with the host tissue.² In this sense, much attention is being paid to scaffolds made of three-dimensional (3D) polymers.⁴ These scaffolds provide the necessary support as artificial extracellular matrices, allowing cells to migrate-proliferate and maintain their differentiated functions. Essentially, they serve as a network to guide the formation of new tissue. In bone tissue engineering, the biodegradable scaffold is a temporary net that is inserted into the defect site or lost bone to initiate bone tissue regeneration, while gradually degrading and being replaced by newly formed bone tissue. An ideal scaffold is characterized by adequate microstructure (pore size and porosity), good biocompatibility and cytocompatibility.^4,5 In addition, it must have the capacity for cell adhesion and retain the metabolic functions of the adhered cells.

There are different materials used in the elaboration of scaffolds for tissue engineering; some are of synthetic origin and others of natural origin, chitosan (Cs) being one of the most important of the latter group. This material is a polysaccharide that is commonly obtained by extensive deacetylation of the chitin of some crustaceans.⁶ Cs is a biopolymer with a wide variety of biomedical applications due to its low or null toxicity properties and its bioactive properties (hemostatic, antimicrobial, cytocompatibility, biocompatibility, etc.). In addition, it is commercially available, at a relatively low cost, and has been widely used to manufacture biomaterials in the pharmaceutical and medical fields in skins, cartilage, and bone tissue regeneration applications.⁷

Carboxymethylcellulose (CMC), is a polyanionic polymer derived from cellulose that has the ability to react with charged molecules within specific pH ranges. Another characteristic that it presents is that the products resulting from the degradation of the scaffold are biocompatible and biodegradable, so it would not have reactions that could cause the rejection of the scaffold once it is implanted.^8,9 CMC has been used in conjunction with some polymers such as PVA for the preparation of supports for bone tissue engineering, however there are few investigations in which it has been used with Cs.^8–10

Now, to focus on bone engineering applications, it was thought to make use of one ceramic component that is a constituent of the bone matrix: Calcium Carbonate (CaCO₃), and in this way simulate to a great extent the extracellular matrix of bone tissue. CaCO₃ is a bioabsorbable bone filling material, which has properties of improved osteoconductivity when it is found as a constituent of the scaffold matrix.^11,12

On the other hand, Zinc oxide (ZnO) is going to be added since it has important properties in the mineralization process,^13,14 as well as a matrix catalyst for its remodeling with an induction of apoptosis of mature osteoclasts.^15,16 These properties will help the properties of the scaffold to speed up the process of bone tissue repair.

The major component to be used in the scaffolds will be Cs, and to make the 3D scaffolds, the chitosan hydrogels can be subjected to a lyophilization process to obtain three-dimensional matrices that can be used for tissue engineering applications, since lyophilization maintains the porous and interconnected structure by removing ice crystals from the frozen chitosan solution.^10,17

Hydrogels can be classified into chemical and physical hydrogels based on their cross-linking mechanism. Chemical crosslinking is a very versatile method for producing hydrogels, but the crosslinking agents used can not only affect the integrity of the entrapped substances (eg, proteins, cells), but are also often toxic compounds that affect the integrity of the cells. Furthermore, the crosslinking agents can cause unwanted reactions with bioactive substances present in the hydrogel matrix. However, this approach requires chemical modification of the primary structure of chitosan, which could affect its initial bioactive and physicochemical properties, particularly if amino groups are involved in the reaction.¹⁸ A widely used approach to generate chemically cross-linked chitosan hydrogels is through the use of glutaraldehyde which reacts with the amino groups of chitosan producing imine bonds.¹⁹ In this way, the concentration of free amino groups of Cs decreases, affecting its biological performance since this group is responsible for the interaction of chitosan with proteins, cells and living organisms.^18,20 This negative effect can be avoided with the use of physical crosslinking, since the hydrogel formation takes place in the absence of organic solvents and toxic crosslinking agents. Physical crosslinks include intertwined chains, hydrogen bonds, and hydrophobic interactions mainly and although they are transient, their characteristics may be sufficient to make hydrogels insoluble in an aqueous medium and can be used in tissue engineering.^10,21,22 An interesting procedure to obtain physically cross-linked hydrogels is that reported by Montembault et al.,²³ who prepared materials by contacting a chitosan solution in an atmosphere of gaseous ammonia. This procedure generates a physical hydrogel that keeps the amino groups of the chitosan structure available and therefore its excellent biological properties are available to be exploited.

That is why this research aims to study an alternative method to form a physical gel (ammonium hydroxide) of Cs-CMC-ZnO-CaCO₃ without using chemical crosslinking compounds, in order not to affect the biocompatibility of the material, as well such as the availability of amino groups (-NH₂) in the Cs molecule, which are generally involved in the chemical crosslinking reaction; In this way, a decrease in the bio-functionality of the designed scaffold would be avoided, to then subject the hydrogel to lyophilize and simulate the bone extracellular matrix with the obtaining of three-dimensional scaffolds with adequate porosity and pore size to evaluate biocompatibility-cytotoxicity properties and osteoinduction in human dental pulp stem cells (hDPSCs) for possible application in bone tissue engineering.

Materials and methods

Materials

Chitosan (Cs) with catalog #448877, carboxymethylcellulose (CMC) with catalog #323063, zinc oxide (ZnO) with catalog #96479 and calcium carbonate (CaCO₃) with catalog #26190 were used in the preparation of the scaffolds. Ammonium hydroxide (HA, 28% NH₃ in H₂O, ≥99.99% trace metals basis, No. 338,818) and glacial acetic acid (AA, ≥99.5% purity No. A6283) were used to make the hydrogels. All solvents and reagents are Sigma-Aldrich brand reagent grade (Toluca, Mexico).

Preparation of solutions

A Cs solution at 2.5% (w/w) was prepared, dissolving in 0.2 m glacial acetic acid at room temperature and stirring for 24 h.

A CMC solution at 3% (w/w) was prepared, dissolving in 0.2 m glacial acetic acid at room temperature and stirring for 24 h.

Composite scaffolds

For the preparation of the scaffolds, the concentrations are Cs (100), Cs-CMC (80/20), Cs-CMC (50/50), Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5), Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5) and Cs-ZnO-CaCO₃ (99/0.5/0.5), (w/w) ratio respectively, with constant stirring for 24 h at room temperature. The polymeric mixture was aliquoted and placed inside a hermetically closed recipient and a beaker containing an excess of ammonium hydroxide was also introduced. Physical cross-linking (gelation) was induced by ammonia diffusion during 24 h.²³ Afterwards, the hydrogels were washed with distilled water in order to remove the ammonium acetate (CH₃COONH₄) and the rest of the ammonium hydroxide that remained in the hydrogels (NH₄OH), until a pH of seven was obtained. To obtain the 3D scaffolds, the hydrogels were placed in a FREEZE DRYER model SCIENTZ-10N at −58°C, and a pressure of 1 Pa for 16 h.²⁴

Physicochemical characterization of 3D scaffolds

Scanning electron microscope

The morphology of the structure of the porous scaffolds was observed by scanning electron microscopy (SEM), using Hitachi SU8230 equipment at 1.0 kV. The scaffolds were submerged in liquid nitrogen to later be cut with a scalpel blade into fine sections. The scaffolds were placed on aluminum tape and covered with a layer of gold using an LLC model to high vacuum Desk II. Similarly, micrographs were used to measure the apparent diameter of the pores of the scaffold structures using Ps© 1990-2021 Adobe software.

Specific surface area

The analysis was carried out by nitrogen adsorption at 77 K to determine the specific surface area of the scaffolds, using Quentachome NOVA 2200e Instruments. The samples were degassed for 12 h, at 95°C, before the measurements to eliminate the air and moisture present in the samples. The Brunauer-Emmett-Teller (BET) model was applied to fit the isotherms and calculate the specific surface area of the analyzed materials.²⁵

Fourier transform infrared spectroscopy

The spectra of the scaffolds were obtained using a Perkin Elmer Spectrum Two FTIR spectrometer. Spectra were analyzed in the wavenumber range of 4000-650 cm⁻¹ with a resolution of 4 cm⁻¹ and a ratio of 100 scans.

Thermal gravimetric analysis

The thermogravimetric study was carried out in a Mettler Toledo TGA/DSC 2 + thermal analyzer, with a nitrogen atmosphere. Scaffolds were measured between 25°C and 600°C with a heating ramp of 20°C/min.

Moisture absorption

Specimens of each scaffold type were dried under reduced pressure for 2 days. Subsequently, the scaffolds were stored under two relative humidity percentages (RH): 50 ± 1% and 91 ± 1%, using magnesium nitrate hexahydrate and sodium sulfate decahydrate, respectively. The mass of each of the samples was taken at the beginning of the study until they reached equilibrium. The percentage of moisture absorption (A. H.) is given by

A (t) = (\frac{m t - m 0}{m o}) 100

(1)

Where m₀ is the mass of the sample at the beginning of the exposure to moisture and m_t is the mass of the sample after a time t in days. The mean and standard deviation of a triplicate for each scaffold were reported.

Optical property

The color of the scaffold surface was determined with a Minolta colorimeter (model CR300, Minolta, Tokyo, Japan), which was calibrated with a standard mosaic (Y = 92.4, X = 0.3162, y = 0.3324) based on the method reported by García et al.,²⁶ The scale used was the CIE-Lab and the parameters measured were luminosity (L*), color parameters a* (red to green) and b* (yellow to blue). The color was measured at three points of different scaffolding, which was previously placed on the standard mosaic. These parameters were used to determine the color difference (ΔE) with

Δ E = \sqrt{{{(Δ L)}^{2} + (Δ a)}^{2} + {(Δ b)}^{2}}

(2)

Where L*₀, a*₀ and b*₀ represent the color parameter values of the standard and L*, a* and b* represent the color parameters of the sample.

Characterization of 3D scaffolds with hDPSCs

Sample sterilization

The samples were sterilized by exposure to UV radiation for 20 min (on each side) using a laminar flow hood (Telstar®, Bio II advance, type Class II).

Alamar blue assay

The cell viability assay was analyzed using the colorimetric Alamar blue™ (Invitrogen, batch #2335658) Cellular Metabolic Assay. The scaffolds were placed in 96-well culture plates and then human dental pulp stem cells (hDPSCs) (The hDPSCs are obtained from pulp tissue, is primary culture. They are routinely worked on in the Tissue Engineering Laboratory according to the protocols requested by the university) (8 × 10⁶) were seeded on the scaffolds, and wells without scaffolds were taken as a control group. Cells were cultured in low glucose Dulbecco’s Modified Eagle Medium (DMEM) (Bio-west, Mexico lot #MS019Y), added with 10% fetal bovine serum (FBS) (Bio-west, Mexico) and 1% penicillin-streptomycin (Sigma-Aldrich). The incubation conditions were at 37°C, in an atmosphere with 5% CO₂ and 95% humidity. At times of 3, 7 and 10 days, the medium was removed, 90 μL of fresh medium and 10 μL of Alamar blue™ were added, th en the cells were incubated for 4 h under the same conditions mentioned. Thecell viability was read at a wavelength of 492 nm in a microplate reader (PKL PPC 142, Pokler Italy). To eliminate the experimental error, three repetitions were made in each of the treatments used in the analysis.

Osteogenic differentiation of hDPSCs on a scaffold

A cell density of 1 × 10⁴ hDPSCs was seeded on the scaffolds in a 24-well plate. Scaffolds were cultured with and without MesenCult™ osteogenic differentiation medium (Stem Cells Technologies, Cambridge, MA, USA), were incubated at 37°C, 5% CO₂ with humidity (BINDER incubator 13–16,721, Germany). The medium was changed twice a week for 28 days. The demeanor was analyzed by alizarin red staining (ARS) techniques and RUNX2 and COL-1 antibody expression.

Evaluation of the osteoinductive properties of scaffolds

Alizarin red staining

The principle for alizarin red S (Sigma Aldrich, USA) is to selectively stain calcium deposits and has recently been used to study calcium-rich deposits formed in cell cultures .²⁷ After 28 days of keeping the scaffolds in a culture medium, fixed with 4% formalin solution for 20 min and then were washed 3 times with 1 mL of PBS-1X each well. Afterwards, the cells fixed on the scaffolds were washed with distilled water to confirm the correct removal of any residual salt. Subsequently, a solution of Alizarin Red S 2% w/v (Sigma Aldrich, USA) was added, with a pH adjusted to 4.2, until the surface of the scaffolds was completely covered. After 1 h incubation at room temperature, excess Alizarin Red siemens was removed by washing with 3 times H₂O. Images of ARS staining were performed using a Leica DM IL LED inverted light field phase contrast light microscope.

Immunocytochemistry

Evaluation of osteogenic differentiation of hDPSCs was analyzed by immunofluorescence. After 28 days of keeping the scaffolds without MesenCult™ osteogenic differentiation medium: Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5), Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5) and Cs-ZnO-CaCO₃ (99/0.5/0.5), they were washed 3 times with 1 mL of PBS-1X each well and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Afterwards, each well was made 2 times with 1 mL of PBS-1X, subsequently the cell constructs were permeabilized with Triton X-100 at 0.1% for 20 min and then washed 3 times with 1 mL of PBS-1X each well and then incubated with BSA at 1% in PBS-1X for 1 h at room temperature. Once again, three washes were performed with 1 mL of PBS-1X each well, and then the cells were incubated overnight at room temperature with primary antibodies RUNX2 (SC390352, Invitrogen, USA) and COL-1 (MA1-26,771, Invitrogen, USA) at a dilution of 1–500 respectively. The expression of antibodies against RUNX2 and COL-1 was analyzed in independent samples in order to analyze the expression of these proteins independently of whether they were revealed with the same fluorophore. The cells were washed 3 times with PBS-1X and then incubated with the secondary antibody Fluorescein isothiocyanate or fluorescein-5-isothiocyanate (Fitc Jackson Immuno research USA), also called FITC for its acronym in English (Fluorescein IsoTioCyanate), diluted in BSA 1–500 and kept at room temperature, protected from light for 1 h. Finally, the wells were washed 3 times with PBS-1X and confocal images were sequentially acquired with a Nikon A1R + laser scanning confocal head coupled to an Eclipse Ti-E inverted microscope (Nikon Corporation, Tokyo, Japan) equipped with a motorized stage (TI-S-E, Nikon) and controlled through Nis Elements C v.5.00 software.

Statistical analysis

Data analysis was expressed as a mean ± standard deviation. Statistical analysis was using a one-way analysis of variance (ANOVA) followed by Tukey’s test. For significant differences, a value of p < 0.05 was taken.

Results and discussion

Characterization of 3D scaffolds

Scaffolds morphology

Figure 1 shows SEM micrographs of scaffolds obtained at 50x (A1-F1) and 100x (A2-F2) magnification. As can be seen (red circles) A2: Cs and B2: Cs-CMC (80–20) present the most homogeneous morphology of all the prepared scaffolds. Now when a higher proportion of CMC is added to the C1: Cs-CMC scaffold (50–50), a heterogeneous morphology between two porosity dimensions begins to be seen (yellow circles and yellow arrows). In contrast, SEM micrographs for scaffolds D: Cs-CMC-ZnO-CaCO₃ (49–50-0.5–0.5), E: Cs-CMC-ZnO-CaCO₃ (79–20-0.5–0.5), and F: Cs -ZnO-CaCO₃ (99–0.5-0.5) showed a completely heterogeneous morphology. ZnO-CaCO₃ was added to these scaffolds and may be related to the presence of these components, seeing these changes in the porous matrix of the scaffold (green and red arrows). In addition, the incorporation of these components to the mixture produced materials with a very different morphology with the presence of entanglements (see green arrows). This indicates that the ZnO-CaCO₃ components could have a strong influence on chitosan polymer chains, which produce three-dimensional structures with this peculiarity. Suggesting for future studies an EDX analysis to corroborate the changes in the morphology of the scaffolds when ZnO-CaCO₃ is added.

Fourier transform infrared spectroscopy

Figure 3 presents the infrared spectra of the scaffolds designed in this research. All the spectra were similar to each other, showing the characteristic absorption bands of Cs; in the wave number range of 3500-3300 cm⁻¹ a wide and intense band was observed, assigning the O-H and N-H stretching vibrations. Furthermore, bands were observed at 1653 cm⁻¹, which is associated with the C = O stretching of amide I, and at 1580 cm⁻¹, related to the deformation of amide II.^1,29,30 Peaks were also detected at 2923 cm⁻¹ and 2880 cm⁻¹, associated with methylene groups and finally, in the range of 1200-1000 cm⁻¹, characteristic signals of the saccharide structure.³¹

The absorption band at 1653 cm⁻¹, shown by the FTIR spectrum of the Cs material, decreased in intensity in those materials where the composition of the scaffolds increased the composition of the CMC (Cs-CMC (50/50) and Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5)), this fact confirms the presence of this component in the scaffold mix.³² While the absorption peak at 1078 cm⁻¹ is attributed to the bending vibration of the Cs stretching group C–O, showing a shift of these peaks with a shift to a higher wave number, indicating the interaction between this group and ZnO.³³ The presence of CaCO₃ in the mixture of the new materials can be seen in the spectra. FTIR data of the developed CaCO₃ composite showed characteristic CaCO₃ peaks at 877 and 1427 cm⁻¹ (-CO₃ stretch) indicating the presence of CaCO₃ in the composites (Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5), Cs-CMC- ZnO-CaCO₃ (49/50/0.5/0.5) and Cs-ZnO-CaCO₃ (99/0.5/0.5).³⁴ The previous results confirm that the mixture of components of the scaffold mimics or simulates an artificial microenvironment to be replaced by an extracellular matrix without any change in the structure of the molecules of the mixture during the preparation treatment of the scaffold.

Thermal gravimetric analysis

Figure 4 presents the TGA analyses for the samples of all the scaffolds prepared in this work. In all the treatments, a loss of mass can be observed at 100°C (red arrow), this corresponds to the assigned loss of water physically adsorbed and weakly hydrogen-bonded to chitosan.^35,36 Scaffolding it can also be seen that Cs, Cs-CMC (80/20), and Cs-CMC (50/50) show a very similar thermal stability, with a maximum mass loss between 300–310°C (blue arrow). This is corroborated by experimental tests from other investigations and corresponds to the presence of Cs and CMC in the designed scaffolds.^24,32,37 Now when ZnO or CaCO₃ are added to the scaffolds (Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5), Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5) and Cs-ZnO-CaCO₃ (99/0.5/0.5)] these show a lower mass loss (black circle in dotted line) than those that do not, because these components are more stable at the decomposition temperatures of Cs and CMC.^38,39 The foregoing corroborates at the end of the test a lower mass loss compared to those that do not present ZnO or CaCO₃.

Moisture absorption

During the experiment at 50% R.H. (Figure 5) the absorption in the scaffolds was 6–10%, in contrast, when the materials were exposed to 91% R.H. absorptions in the range of 38–50% were obtained. Therefore, it is shown that the designed scaffolds have a hydrophilic character when induced at high R.H. This characteristic can be used since it will have a great capacity for H₂O absorption, similarly when we talk about bioactive tests it will also be very useful since it will allow the absorption of the culture medium during cell proliferation. This moisture absorption can be related to the availability of the amino group in the Cs molecule, since it is the component in greater proportion that makes the scaffold hydrophilic.⁴⁰

Pore size distribution

The Figure 2 shows the pore size distribution of the scaffolds prepared in this research work. Also, it is shown in Table 1 The averages of the pore sizes of the different elaborated scaffolds. In general, all samples were found to have a unimodal distribution. It should be noted that the morphology of the Cs samples presents a distribution in terms of pore dimensions with symmetrical characteristics, with values in the highest number of repetitions between the range of 30–76 μm. Such pore dimensions are present in most of the treatments except for Cs-ZnO-CO3 (99/0.5/0.5) where the pore sizes are above 76 μm. The rest of the materials Cs-CMC (80/20), Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5), Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5) and Cs-ZnO-CaCO₃ (99/0.5/0.5) exhibited an asymmetric distribution to the left with values in greater repeatability of pore size between 50-80 μm, while for ZnO-CaCO₃ (99/0.5/0.5) they were between 100-130 μm. On the other hand, the Cs-CMC scaffold showed an asymmetric distribution with a slight bias to the right, with a higher repetitive number between the ranges of 76–100 μm. The scaffolds must have the characteristic of being porous to allow cell ingrowth and allow proliferation. The dimensions obtained in the investigation are very similar to studies where they demonstrated that the minimum pore size for significant bone growth is 75–100 μm with an optimal range of 100–135 μm.²⁸ This suggests that our materials are in appropriate ranges for possible use in applications in bone tissue engineering.

Table 1.

Average pore size (T_p) of 3D-materials.

Material	T_p (μm)^a
Cs	56.0 ± 13.3
Cs-CMC (80/20)	62.3 ± 18.6
Cs-CMC (50/50)	82.4 ± 18.6
Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5)	83.7 ± 18.7
Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5)	88.6 ± 13.13
Cs-ZnO-CaCO₃ (99/0.5/0.5)	134.3 ± 27.1

^aAverage value from 50 measurements.

Optical property

The optical properties (Table 2) of the mixtures of the scaffolds with the Cs/CMC/ZnO/CaCO₃ components and the Cs-CMC (50/50) scaffold are the ones that showed an increase in the luminosity values (L*), concerning those materials with Cs and low CMC content (Cs-CMC (80/20). The opposite demeanor, was in the observed parameter ΔE where the increase was in the materials without ZnO/CaCO₃ and low content of CMC. Regarding the parameter a* (redness/greenness) where the components containing ZnO and CaCO₃ show significant differences with respect to the scaffolds that did not contain it. A different demeanor occurred in the parameter b* (yellowness) where the presence of a higher proportion of Cs shows an increase in the values, which gives it a color with an incidence of yellow, demeanors similar to those reported by other researchers.⁴¹

Table 2.

Optical properties of the formulated scaffolds.

Andamio	L*	a*	b*	ΔE
Cs	83.1 ± 3.6^b	−1.5 ± 0.2^b	6.9 ± 0.8^e	15.6 ± 0.5^d
Cs-CMC (80/20)	80.7 ± 3.7^a	−1.1 ± 0.3^a	4.0 ± 0.5^cd	17.2 ± 0.9^e
Cs-CMC (50/50)	85.2 ± 0.9^c	−1.1 ± 0.3^a	2.5 ± 0.4^a	12.5 ± 0.3^b
Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5)	85.6 ± 2.1^c	−1.4 ± 0.1^b	3.3 ± 0.9^b	12.3 ± 0.2^ab
Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5)	87.2 ± 0.7^d	−1.5 ± 0.1^b	3.6 ± 0.8^bc	10.8 ± 0.1^a
Cs-ZnO-CaCO₃ (99/0.5/0.5)	80.9 ± 2.6^a	−1.5 ± 0.1^b	4.4 ± 0.4^cd	17.1 ± 0.9^e

Values are given of n = 3 ± SD.

^{a, b, c, d, e} in the same column indicate a significant difference (p < 0.05).

Biological characterization of 3D scaffolds

Alamar Blue^MT assay

There are many methods to evaluate the cytotoxic effect of scaffolds on cultured cells by monitoring non-specific alterations in basic cell functions such as mitochondria, plasma membrane integrity, etc.⁴² One of them is the cell metabolic Alamar blue^TM, where the results of cell viability are shown in Figure 6, it was analyzed by ANOVA one way, p < 0.05, Tukey’s test. Regardless of the chosen assay, a material is considered as not cytotoxic effect when it yields cell viability results above 70%.²⁴

There are significant differences between the materials that were manufactured only with Cs and the rest of the treatments. Likewise, differences are observed between the materials in which the components of their matrix were Cs-CMC in relation to those in which ZnO and CaCO₃ were added(Cs-CMC-ZnCaCO₃ (79/20/0.5/0.5) and Cs-CMC- ZnCaCO₃ (49/50/0.5/0.5)). However, there were no significant differences with the scaffold that did not present CMC (Cs-ZnO-CaCO₃ 99/0.5/0.5). These results are comparable to those of Chernozem et al., (2019) since they incorporate CaCO₃, the cell viability results are above 80%.⁴³ A very similar demeanor is found in scaffolds containing CMC and CaCO₃ added.⁴⁴ Other research obtains very similar viability values by incorporating ZnO into their scaffolds, which is why ours are showing demeanor above 85%.⁴⁵ These data make us think that the designed materials can be applied in tissue engineering.

Osteogenic differentiation of hDPSCs on a scaffold

The hDPSCs seeded on the different 3D scaffolds with or without MesenCultTM osteogenic differentiation medium are shown in Figure 7. The results show calcium deposits for the scaffolds Cs-CMC-ZnO-CaCO3 (79/20/0.5/0.5), Cs-CMC-ZnO-CaCO3 (49/50/0.5/0.5) and Cs-ZnO-CaCO3 (99/0.5/0.5). These results are compared with those obtained by Laurenti end Cauda (2017) whereby incorporating ZnO into the scaffolds, calcium deposits are increased due to the increase in the active sites of the scaffold that could promote the interaction of osteoblast proteins to lead to a production of calcium deposits.⁴⁵ Our results agree with those reported by Gong et al. (2019) although they did the histological staining in the well and we did the scaffold., therefore we can relate CaCO₃ as a material that provides the scaffold with osteoinductive properties in stem cells.⁴⁶

Immunofluorescence by RUNX2 and COL-1 of hDPSCs on scaffolds

Few studies have worked with the safe and effective incorporation of CaCO₃ in scaffolds as bone substitutes as a constituent to stimulate cell proliferation and differentiation.^46–48 Figure 8 shows the osteoinductive properties of scaffolds containing CaCO₃, the expression of markers RUNX-2 and COL-1 representative of osteogenic differentiation was evaluated. Being able to observe that the constructs of Cs-CMC-ZnO-CaCO₃ (79/20/0.5/0.5), Cs-CMC-ZnO-CaCO₃ (49/50/0.5/0.5) and Cs-ZnO-CaCO₃ (99/0.5/0.5) showed expression for both the RUNX-2 and COL-1 markers. Our results agree with investigations where the materials in which CaCO₃ was added to the polymeric matrix of the scaffolds provide them with the characteristic of the expression power of RUNX-2 and COL-1 markers.^43,49 The COL-1 expression of osteogenic lineage differentiation for the Cs-CMC-ZnO-CaCO₃ (8-B₁) scaffold was seen with slightly lower expression and this is caused by the higher concentration of CMC, since when it is present, it causes a lower disposition of CaCO₃ due to greater availability of polymeric chains of the CMC molecules, which allows a slow differentiation. The same demeanor was shown in the research of Huang et al., (2020) where a better proliferation and adhesion is observed with a slower differentiation in the materials that contain CMC, instead when the presence of CaCO₃ is found, the cells tend to differentiate instead of proliferating and therefore there is less expression.⁵⁰

Conclusions

The scaffolds designed in this research work where the gel formation technique was used that was stabilized by physical bonds and subsequently dried by the lyophilization technique to obtain the three-dimensional geometry, presented morphological characteristics with pore size distributions within the scaffolds, dimensions necessary to promote cell proliferation. The results of the physicochemical characterizations of FTIR and TGA confirmed the presence of the polymeric components in the matrices of the elaborated scaffolds. The moisture absorption test showed that the elaborated scaffolds were hydrophilic in nature, which promotes the water absorption capacity that is essential for the management of cell cultures. The optical properties showed us similar values for the materials with Cs that we designed and corroborates that no chemical modification was suffered in the chemical chains of the polymeric components worked on. Regarding the biological characteristics of the scaffolds, they showed that there is no cytotoxic effect for any scaffold at the different evaluation times, analyzed with the Alamar blue^TM cellular metabolic assay. The evaluation of the differentiation to osteogenic lineage shows that the incorporation of CaCO₃ in the scaffolds gives it osteoinductive properties in the stem cells. All these evaluations showed favorable results that increase its possible application in bone tissue engineering.

Footnotes

Acknowledgements

We thank Dr Rita Sulub-Sulub for her support in the TGA tests. The authors acknowledge Dr Jesús Madera-Santana for his support in carrying out the tests on optical properties and stability of the designed scaffolds. We thank Dr Miguel Tapia (Unidad de Microscopía, IIBO-UNAM).

Author contributions

All authors have contributed equally to the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Program for Research and Technological Innovation Projects (PAPIIT) UNAM. Grant: IA207420 and by the DGAPA department, UNAM for the Postdoctoral Scholarship.

ORCID iDs

Raúl Rosales-Ibáñez

Reyna-Urrutia Alonso VA

Miriam Estevez

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Biological activity of a chitosan-carboxymethylcellulose-zinc oxide and calcium carbonate in 3D scaffolds stabilized by physical links for bone tissue engineering

Abstract

Keywords

Introduction

Materials and methods

Materials

Preparation of solutions

Composite scaffolds

Physicochemical characterization of 3D scaffolds

Scanning electron microscope

Specific surface area

Fourier transform infrared spectroscopy

Thermal gravimetric analysis

Moisture absorption

Optical property

Characterization of 3D scaffolds with hDPSCs

Sample sterilization

Alamar blue assay

Osteogenic differentiation of hDPSCs on a scaffold

Evaluation of the osteoinductive properties of scaffolds

Alizarin red staining

Immunocytochemistry

Statistical analysis

Results and discussion

Characterization of 3D scaffolds

Scaffolds morphology

Fourier transform infrared spectroscopy

Thermal gravimetric analysis

Moisture absorption

Pore size distribution

Optical property

Biological characterization of 3D scaffolds

Alamar BlueMT assay

Osteogenic differentiation of hDPSCs on a scaffold

Immunofluorescence by RUNX2 and COL-1 of hDPSCs on scaffolds

Conclusions

Footnotes

Acknowledgements

Author contributions

Declaration of conflicting interests

Funding

ORCID iDs

References

Alamar Blue^MT assay