The Biocompatibility of Multi-Source Stem Cells and Gelatin-Carboxymethyl Chitosan-Sodium Alginate Hybrid Biomaterials

Abstract

Background:

Nowadays, biological tissue engineering is a growing field of research. Biocompatibility is a key indicator for measuring tissue engineering biomaterials, which is of great significance for the replacement and repair of damaged tissues.

Methods:

In this study, using gelatin, carboxymethyl chitosan, and sodium alginate, a tissue engineering material scaffold that can carry cells was successfully prepared. The material was characterized by Fourier transforms infrared spectroscopy. In addition, the prepared scaffolds have physicochemical properties, such as swelling ratio, biodegradability. we observed the biocompatibility of the hydrogel to different adult stem cells (BMSCs and ADSCs) in vivo and in vitro. Adult stem cells were planted on gelatin-carboxymethyl chitosan-sodium alginate (Gel/SA/CMCS) hydrogels for 7 days in vitro, and the survival of stem cells in vitro was observed by live/died staining. Gel/SA/CMCS hydrogels loaded with stem cells were subcutaneously transplanted into nude mice for 14 days of in vivo culture observation. The survival of adult stem cells was observed by staining for stem cell surface markers (CD29, CD90) and Ki67.

Results:

The scaffolds had a microporous structure with an appropriate pore size (about 80 μm). Live/died staining showed that adult stem cells could stably survive in Gel/SA/CMCS hydrogels for at least 7 days. After 14 days of culture in nude mice, Ki67 staining showed that the stem cells supported by Gel/SA/CMCS hydrogel still had high proliferation activity.

Conclusion:

Gel/SA/CMCSs hydrogel has a stable interpenetrating porous structure, suitable swelling performance and degradation rate, can promote and support the survival of adult stem cells in vivo and in vitro, and has good biocompatibility. Therefore, Gel/SA/CMCS hydrogel is a strong candidate for biological tissue engineering materials.

Graphical Abstract

Introduction

Tissue engineering is the new direction of current medical development. The rapid development of tissue engineering provides a green channel for alleviating organ shortages, promoting damaged tissue repair, and maximizing patients’ quality of life [1, 2]. The biomaterial scaffold is a key component of tissue engineering. The biomaterial used for tissue engineering must meet specific requirements [3]:

1. To support cell attachment, migration, cell–cell interaction, cell proliferation and differentiation.

2. The host immune system into the engineered tissue is biocompatible.

3. Biodegradation at a controlled rate matches the growth rate of new tissues and promotes the integration of the engineered tissue with surrounding host tissues.

4. In the initial stage after implantation, provide structural support for cells and new tissues formed in the scaffold.

After being dissolved in water, sodium alginate cross-links with the non-cytotoxic concentration of divalent cations (Ca²⁺) to form a gel at room temperature [4, 5]. However, the three-dimensional space formed inside the pure sodium alginate hydrogel is relatively fragile, and it is difficult to provide stable growth space for cells for a long time [4–6]. This shortcoming limits its application in biological tissue engineering technology. Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, contains COOH and NH₂ groups in the molecule, soluble in neutral and alkaline aqueous solutions [5]. Carboxymethyl chitosan does not react chemically with sodium alginate. The cross-linking between the two is achieved through the bond between the amine group of carboxymethyl chitosan and the carboxyl group of sodium alginate. Compared with the sodium alginate hydrogel, carboxymethyl chitosan provides the hydrogel with more substantial mechanical properties [4, 5, 7]. It can provide a more stable three-dimensional structure for the survival of cells.

Another critical component of tissue engineering is the “seed cell”, cell-based tissue engineering requires a reliable source of cells. Stem cells have become the preferred seed cells for tissue repair due to their multi-directional differentiation potential and self-renewal ability [8]. According to the developmental stage and the source of differentiation, stem cells can be divided into embryonic stem cells (ESC) and adult stem cells (ASC) [2, 3]. Recent advances in the use of adult stem cells have in part allowed these stem cell types to apply in cell-based tissue engineering as possible sources.

In this study, we studied the performance of a network cross-linked polysaccharide hydrogel in vivo and in vitro histocompatibility performance (Fig. 1). This cross-linked polysaccharide hydrogel is composed of gelatin, carboxymethyl chitosan and sodium alginate. The amino group of carboxymethyl chitosan can quickly crosslink with the hydroxyl group of sodium alginate to form a network hydrogel. The electrostatic interaction between gelatin and polysaccharide further stabilizes the structure of polysaccharide hydrogel. Carboxymethyl chitosan has excellent water solubility, antibacterial properties and biocompatibility. The amine group of carboxymethyl chitosan and the carboxyl group of sodium alginate cross-linking to form a stable and uniform spatial structure provides a spatial prerequisite for cell survival [5].

Fig. 1.

Experimental flowchart

Materials and methods

Materials

CaCl₂ (MW ~ 110.98 Da) and sodium alginate (MW ~ 240 kDa) were obtained from Yuanye Biological Technology (Shanghai, China). Chitosan (MW ~ 200 kDa) was purchased from BoMei Biotechnology Co.Ltd (Hefei, China). Carboxymethyl chitosan (MW ~ 230 kDa) and gelatin (MW ~ 200 kDa) were purchased from Solarbio Science & Technology (Beijing, China). BALB/c-nu mice and SD rats were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd (Jinan, China).

Biomaterial preparation

The gelatin (concentration of 0.1 g/mL), sodium alginate (concentration of 0.01 g/mL), carboxymethyl chitosan (concentration of 0.02 g/mL) and CaCl₂ (concentration of 2%) were dissolved into deionized water (R = 18.2 MΩ/cm) under constant stirring at 50 °C [5], respectively. The filters (Jinteng, China), which specifications are 0.8 nm, 0.45 nm and 0.22 nm in order, were used to filter the above solution to make them sterile. Then, Gel/SA/CMCS hydrogel was prepared in a ratio of 1:1:1. The resultant Gel/SA/CMCS hydrogel was then ionically cross-linked by 2% CaCl₂ solution for 10 min. SA/CMCS hydrogel was prepared in the same way but without gelatin.

FTIR spectroscopy

An Avatar 360 FTIR spectrometer (Nicolet, USA) was utilized to characterize FTIR spectra of the samples (including SA, CMCS, SA/CMCS, Gel/SA/CMCS) over the range of 500–4000 cm⁻¹ at a scanning resolution of 2 cm⁻¹ during 32 scans.

Scanning electron microscopy

The Gel/SA/CMCS hydrogel were frozen in liquid nitrogen and freeze-dried for 24 h at -60 °C (Heto PowerDry PL3000). The dried material was coated with gold and mounted for analysis. Pore size and interconnections between pores were assessed using SEM (ULTRA PLUS, Carl Zeiss, Oberkochen, Germany).

Swelling ratio measurement

A gravimetric method was carried out to analyze the swelling ratio. A known weight of lyophilized simples, in triplicate, was immersed in PBS at 37 °C for swelling. Subsequently, the weight of the swollen sample was measured every 1 h until the weight of the hydrogels no longer increased. The swelling ratio (SR) was calculated by using the formula below (1):

$$SR=\frac{Ws-Wd}{\mathit{Wd}}$$ 1

where Ws and Wd represent the weights of the swollen and dried samples, respectively.

In vitro degradation assay

The degradation behaviour of the simples (i.e., SA, SA/CMCS, Gel/ SA/CMCS) was tested by monitoring the weight changes after 5, 7, 10, and 14 days. The samples (N = 3) were incubated in DMEM containing 10% fetal bovine serum at 37 °C. The samples were removed from the medium at each time point, then dried, and weighted. The degradation ratio (DR) was calculated by using the formula below (2):

$$DR=\frac{Wt-Wd}{\mathit{Wd}}$$ 2

where Wt and Wd represent the weights of the swollen and dried samples, respectively.

Cell isolation, culturing and identify

All animal experiments were conducted in the Central Laboratory of Affiliated Hospital of Jining Medical University, according to the guidelines of the Institutional Animal Care and Use Committee. ADSCs and BMSCs were separated from the groin and femur of SD rats according to previous methods, respectively. In short, under anaesthesia, adipose tissue was collected aseptically from the subcutaneous fat in the groin of 2-week-old SD rats; after the femoral shaft was taken out, the bone marrow cavity was rinsed with 3 ml PBS, and the washing fluid was collected. Adipose tissue was identified by oil red O staining. After the adipose tissue is minced, digested with EDTA (Solarbio), neutralized, centrifuged, and resuspended, it is planted in a 25 ml culture flask; the bone marrow washing solution is lysed twice with red blood cell lysis solution (Solarbio) and inoculated in 25 ml culture flask. Both cells were incubated in DMEM containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C and 5% humidified CO₂. After 48 h, unattached cells were removed by washing with PBS. Change the medium every 72 h until the cells are confluent. When the cells reach 80 ~ 90% confluence, the subculture will continue until the third generation.

ADSCs and BMSCs were seeded on the 96-well plates with a density of 1 × 10⁴ cells/well and incubated in the conditions mentioned above. The cell proliferation was investigated using cell counting kit-8 (CCK-8, Absin Bioscience Inc., Shanghai, China) following the instructions. After incubated for 12 h, 24 h, 48 h, 72 h and 96 h, the CCK-8 mixed medium was transferred into 96-well plates and measured by a microplate reader (SynergyH1/H1M, BioTek, Beijing, China) with 450 nm optical density (OD value).

The specific surface antigens of ADSCs and BDSCs were characterized by flow cytometry. Cells at Passage 3 were trypsinized and obtained a single‐cell suspension. The suspension was labelled with the following fluorescence‐conjugated antibodies: CD90‐PE (Biolegend, San Diego, CA, USA), CD29‐PE (Biolegend, US), CD34‐PE (Abcam, Cambridge, UK).

Cell seeding

Hydrogels were prepared as described above, and 1 ml of cell suspension of ADSCs was mixed at 1 × 10⁵/ml in the hydrogel before ionically cross-linked with 2% CaCl₂. The hybrid hydrogel was evenly spread in a medium dish (60 mm, Corning, Corning, NY, USA) and cross-linked with 2% CaCl₂ for 10 min. 5 ml of culture medium was added and incubated at 37 °C with 5% humidified CO₂ after removing the excess liquid. The medium was changed every two days. BDSCs were treated in the same way.

The ADSCs-laden Gel/SA/CMCS hydrogels were fixed with 2% glutaraldehyde for 30 min at room temperature, washed with water twice, and then soaked in 30%, 50%, 70%, 80%, 90% and 100% alcohol for 10 min for gradient dehydration, respectively. After immersed in tert-butanol twice for 5 min, the ADSCs-laden hydrogels were lyophilized for SEM analysis [9].

Hybrid material cytotoxicity detection

To investigate the cytotoxicity of all materials, 20 ml of sterilized hydrogels was immersed in 50 ml of the complete medium at 37 °C for 2 days. ADSCs were seeded on the 96-well plates with a density of 1 × 10 4 cells/well. The solid in the medium was discarded, and the remaining liquid was employed for cell culture in 96-well plates. The proliferative activity of the cells was determined by the same method as above after incubating for 1,2,3,4 and 5 days.

Live/dead cell assay

Calcein AM and propidium iodide (PI) were used to identify the cell viability of cells-planted in hydrogels. The kit (Invitiogen, China) was a double-staining procedure using Calcein AM and PI at 1,3,5 and 7 days. As follows, cells-hydrogel were incubated with Calcein AM (1 μM) at 37 °C for 30 min and then washed by PBS three times. Then the scaffolds were incubated with PI (1 μM) under the same conditions for 30 min and washed with PBS three times. A confocal microscope (LSM 800, Carl Zeiss) was employed to obtain the 2D and 3D images. The cell viability at 3, 5, and 7 days was calculated using Image J software.

Implantation of cell-seeded hydrogel in vivo

The cell-seeded hydrogels were implanted subcutaneously into 8-week-old male athymic mice under aseptic conditions. The cell-seeded grafts, 1cm², 0.2 mm thickness, were implanted into each mouse’s right and left side (n = 3). As a control, unseeded hydrogels were planted in an identical fashion (n = 3). The skin incision was closed with 6–0 Ethilon suture material.

Immunocytochemistry

The grafts were harvested 7 and 14 days after implantation, fixed in paraformaldehyde solution (Yuanye, China), dehydrated and embedded in paraffin. Cellular infiltration and the number of cell layers in each graft were assessed using hematoxylin and eosin staining and Masson’s trichrome staining on 5 mm sections. The fluorescent labels of stem cell-specific antibodys (CD29, CD90) were utilized to identify cell types. Recombinant Anti-Ki67 antibody was used to detect cell proliferation activity.

Statistical analysis

All data are shown as the mean ± standard derivation (SD). Statistical analysis was carried out using GraphPad Prism 7 software. Statistical comparisons were performed using one-way analysis of variance (ANOVA) with Bonferroni post-hoc tests and Students’t-tests. Differences were considered statistically significant at p < 0.05.

Results

Characterization of Gel/SA/CMCS hydrogels

Figure 2 displays the FTIR spectra of polysaccharides (SA, Chitosan and CMCS) and their composite hydrogel (Gel/SA/CMCS) to identify the functional groups of various compositions and determine their interactions in the composite hydrogel. Figure 2A shows the peaks of the characteristics of chitosan, the peaks at 2922 cm⁻¹, 1594 cm⁻¹, 1154 cm⁻¹, and 1096 cm⁻¹, can be attributed to the C–H stretching, N–H bending, bridge-O-stretching, and C–O stretching modes of chitosan, respectively [5]. The symmetric and asymmetric stretching vibration of the COO-group of carboxymethyl chitosan were shown in Fig. 2B. Compared with the peak of sodium alginate (Fig. 2C), the peak of Gel/SA/CMCS polymer at 3409 cm⁻¹ was significantly weakened (Fig. 2D), indicating that the carboxyl group of sodium alginate and the amine group of chitosan were successfully combined. The polymers were prepared via an electrostatic interaction and Ca²⁺ crosslinking, which could avoid affecting the physicochemical properties of each component.

Fig. 2.

FT-IR spectra of A Chitosan, B CMCS, C SA and D Gel/SA/CMCS

Figure 3A, B shows Gel/SA/CMCS hydrogel and SA/CMCS hydrogel under normal conditions and after freeze-drying. In the interior of the Gel/SA/CMCS hydrogel, highly interconnected irregular porous structures with relatively uniform size (about 80 μm) were observed, as evidenced by the scanning electron microscopy (SEM) image (Fig. 3C). The porous structure is mainly formed by CMCS and SA cross-linking to form a composite gel whole.

Fig. 3.

Hydrogel material of A Gel/SA/CMCS hydrogels: the top are gel state and the bottom are freeze-dried and B SA/CMCS hydrogels: the top are gel state and the bottom are freeze-dried, C SEM images of Gel/SA/CMCS hydrogels: The inside of the hydrogel has a highly interconnected irregular porous structure (average size: 40 μm), D swelling ratios and E degradation ratios of SA hydrogels, SA/CMCS hydrogels and Gel/SA/CMCS hydrogels: SA/CMCS and Gel/SA/CMCS hydrogels show similar swelling properties and degradation rates

Furthermore, we investigated the swelling properties of the hydrogels. Figure 3D shows the swelling properties of different polymers, and found that all polysaccharide polymers have good swelling properties. It suggests that all the hydrogels can achieve the swelling balance within 5 h. The swelling performance of Gel/SA/CMSC composite hydrogel is significantly lower than that of SA, but it is not significantly different from SA/CMCS.

In this study, we used cell culture media to simulate the microenvironment for cell growth in vivo and observe the degradation efficiency of different hydrogels. As shown in Fig. 3E, under the effect of the in vitro cell culture medium, the Gel/SA/CMCS hydrogel has the lowest degree of explanation within 14 days, and the SA hydrogel has the highest degree of degradation.

Characterization of ADSCs and BDSCs

The primary ADSCs exhibited spindle-shaped cells after being cultured in vitro for 24 h. The third passage cells with 80–90% fusion appeared as monolayers of flat fibroblast-like cells under microscopy (Fig. 4A). The cell morphology of BMSCs is similar to ADSCs (Fig. 4B). As shown in Fig. 4C, flow cytometry results suggested that both third-generation cells expressed stem cell-specific antibodies CD29 and CD90 and were negative for CD34.

Fig. 4.

Cell images of A ADSCs and B BMSCs, and C Flow cytometry of ADSCs and BMSCs

Characterization of Cell-seeded Gel/SA/CMCS hydrogels and toxicity

Figure 5A shows the morphology of Cell-seeded Gel/SA/CMCS hydrogels. It can be seen that the cells adhere to the hydrogel, indicating that they are scattered and evenly distributed. Using the proliferation activity of ADSCs to judge the effect of hydrogel materials on cell growth and proliferation, the results showed that the cell proliferation phase of the experimental group occurred earlier than that of the blank control group, which proved the cell proliferation activity of the experimental group medium (soaked in the hydrogel) enhancement (Fig. 5B, C).

Fig. 5.

A SEM images of cell-seeded Gel/SA/CMCS hydrogels, toxicity performance of B SA hydrogels and C Gel/SA/CMCS hydrogels, A: Hydrogel soaking for 2 days, B: Blank control: the adult stem cells cultured in the complete medium of the hydrogel soaking liquid enter the rapid proliferation mode on the third day after culture (p < 0.05)

Live/dead assay

The viability of the ADSCs and BDSCs embedded within the Gel/SA/CMCS scaffold were evaluated by the Live/Dead assay. As showed in Fig. 6A, both the ADSCs and BDSCs encapsulated within the scaffold maintained high cell viability after 7 day of culture. Image J software was utilized to statistically analyze the cell numbers of the two kinds of adult stem cells in the gel/SA/CMCS hydrogel at 3,5 and 7 days. As shown in the figure, there is no significant difference between the two kinds of cells in vitro culture (p > 0.05), indicating the Gel/SA/CMCS hydrogel has good histocompatibility for stem cells and can provide a reliable attachment place and growth space for adherent cells (Fig. 6B).

Fig. 6.

3D images of A Live/died staining and B Fluorescence semi-quantitative analysis

Histological analysis in vivo

In order to confirm the superiority of Gel/SA/CMCS hydrogel as a tissue engineering biomaterial for histocompatibility, ADSCs-seeded Gel/SA/CMCS hydrogel and BMSCs-seeded Gel/SA/CMCS hydrogel were transplanted separately into the nude mice subcutaneous for culture experiments in vivo. Figure 7 shows the H&E staining result of the hydrogel transplanted in vivo. 7 days after transplantation, the boundary between the hydrogel and the surrounding subcutaneous tissue was clear, and a small amount of inflammatory cell infiltration and fibroblast migration were observed. The stem cells in ADSCs-seeded Gel/SA/CMCS hydrogel and BMSCs-seeded Gel/SA/CMCS hydrogel are relatively evenly distributed. By the 14th day, the boundaries of the hydrogel were still clearly identifiable, and the number of inflammatory cells was increased compared to before. What is more remarkable was the formation of obvious staggered tissue strands and uneven distribution of stem cells in the cell-seeded hydrogel. Most of them are distributed along the cords of new tissues. Figure 8 presented the results of Masson’s trichrome staining. As time went by, all groups showed more and more collagen deposition. Compared with the blank control group, the collagen deposition in the Cell-seeded hydrogel was more obvious.

Fig. 7.

H&E staining images of grafts in vivo including Blank, ADSCs-seeded Gel/SA/CMCS hydrogels and BMSCs-seeded Gel/SA/CMCS hydrogels: The red arrow points to the hydrogel, the black arrow points to inflammatory cells, the green points to planting cells

Fig. 8.

Masson’s staining images of grafts in vivo including Blank, ADSCs-seeded Gel/SA/CMCS hydrogels and BMSCs-seeded Gel/SA/CMCS hydrogels

Subsequently, the sections were further subjected to immunofluorescence staining with stem cell surface markers CD29 (Fig. 9A), CD90 (Fig. 9B) and Ki67 (Fig. 10). Ki67 staining is to further illustrate the proliferation activity of stem cells, and the results showed that the stem cells in the Gel/SA/CMCS hydrogel can grow normally in vivo. This proves that within 14 days of in vivo culture, both types of adult stem cells can survive normally, providing a prerequisite for stem cells to exert effective therapeutic effects in the body.

Fig. 9.

A CD29 and B CD90 staining images of grafts in vivo including Blank, ADSCs-seeded Gel/SA/CMCS hydrogels and BMSCs-seeded Gel/SA/CMCS hydrogels

Fig. 10.

Ki67 staining images of grafts in vivo including Blank, ADSCs-seeded Gel/SA/CMCS hydrogels and BMSCs-seeded Gel/SA/CMCS hydrogels

Discussion

Over the past decades, many of the synthetic biomaterials that have been used in tissue engineering, notably collagen-based materials and the polylactic, polyglycolic, and polycaprolactone family of polymers, were already well known in the medical community, having already been employed as bioresorbable sutures [10–13]. But they were far from optimal for many tissues engineering purposes, mainly because the hydrolytic biodegradation process releases acid, which can be toxic to cells [10, 12]. Polysaccharides are natural compounds that are abundant in nature and have low toxicity [4, 5, 14]. They are widely used in drug delivery and biomedical research [15, 16]. Polysaccharides are derived from algae (alginate and carrageenan), plant sources (cellulose, pectin and guar gum), microbial sources (glucan and chitin) and animal sources (hyaluronic acid, chondroitin and heparin) [17]. Marine polysaccharides not only have good biodegradability and cell compatibility, but also have inherent (bio)physical and chemical functions. In particular, sodium alginate has the advantages of cost-effectiveness, high water absorption, biocompatibility and easy processing, making it widely used in biomedicine and tissue engineering technology [18]. Chitosan is mainly derived from chitin. Chitosan and its derivatives have good antibacterial properties and have become a hotspot in biomedicine and biopharmaceutical research [19, 20]. They are used in tissue engineering, wound healing and as a drug delivery adjuvant [21, 22]. The natural polysaccharide SA is a biologically inert polymer, lacking RGD molecules (responsible for cell attachment), and cells are not easy to adhere to grow. Ca²⁺ can cross-link with the COO- groups of SA to form a single network hydrogel [23]. Interpenetrating networks (IPNs) are created by a combination of two or more distinct hydrogel systems, and it can provide more adhesion sites for cells [24]. The interaction between the carboxyl group of sodium alginate and the amino group of chitosan makes Gel/SA/CMCS hydrogel form a complex network structure, which is helpful for the attachment and growth of cells. The scaffold’s three-dimensional porous structure facilitates the efficient exchange of oxygen, nutrients, and wastes for the encapsulated cells [25]. What’s more, the interconnected pores made the hydrogels own a large specific surface area, which furthers the cells to adhere to the wall.

The equilibrium swelling rate reflects the internal cross-linking degree and structural characteristics of the hydrogel. During the swelling process of the hydrogel, water first penetrates the hydrogel network to expand its volume. But as the water molecules continue to penetrate, the cross-linked network will shrink sharply to prevent water from continuing to enter the hydrogel, resulting in swelling equilibrium [7]. Appropriate swelling ability can make the biological material absorb the exudate from the injury site and promote the healing of the injured tissue [26]. In the entire degradation process, Gel/SA/CMCS hydrogel degradation is divided into two stages. The degradation rate of the first stage is relatively gentle, which may be related to the hydrolysis of the hydrogel. As the cross-links between SA and CMCS dissociating, the degradation rate of the hydrogel increases. Cell-based tissue engineering biomaterials need to have a certain degree of degradability, providing adhesion scaffolds for cells in vitro and complete cell transfer mediators in vivo. Gel/SA/CMCS hydrogel is inert in its early degradation ability, and can later provide a stable space structure and adhesion site for cell culture in vitro; with the dissociation of cross-linked bonds, the degradation rate increases, which releases cells and their active organisms in vivo Factors provide convenience [7, 27]. The line graph drawn according to the experimental data collected by us shows that the adult stem cells cultured in the complete medium of the hydrogel soaking liquid enter the rapid proliferation mode on the third day after culture, which is earlier than the control group (normal complete medium) one day. And statistical analysis showed that the difference was statistically significant between the experimental group and the control group (p < 0.05). This promotion may be related to the dissolution of some natural polysaccharides in the medium and acting on the cells [28–31]. Jin Wang et al. have similar findings in the study of the effects of chitosan derivatives on the proliferation of mesenchymal stem cells, but they did not provide further explanation [32]. The micro-environment of cell growth is affected by elemental ions released from the scaffold extracts. The behavior of cells in tissues and organs is not only regulated by gene sequences and proteins, but also Regulation of external environmental factors [33]. Cell proliferation is mediated by various proteins involved in intracellular calcium signal transduction. Ca2 + channels are involved in normal and pathological cell proliferation. In particular, normal cells require high external calcium concentrations [34].

Transplantation is the most common way to realize the application value of Cell-based tissue engineering. Gel/SA/CMCS hydrogel as a cell carrier can maintain the activity of adult stem cells for at least 7 day, providing time guarantee for its in vivo application. But when we cultured Gel/SA/CMCS hydrogels loaded with BMSCs in vitro, we found that the number of BMSCs decreased on the fifth day. According to our previous experience in cultivating BMSCs and ADSCs, BMSCs culture conditions are more stringent than ADSCs. Even in the same culture environment, they are easily affected by the surrounding environment during the culture process (such as cell exchange time, medium formula, etc.). This may be the reason for this result.

Our results show that Gel/SA/CMCS hydrogel loaded with adult stem cells can promote the repair of damaged tissues. CMCS has certain antibacterial properties [35, 36], because the -NH³⁺ group of chitosan can generate static electricity with the negative charge on the surface of microbial cells [37], which can destroy the bacterial wall to a certain extent to achieve antibacterial effect [38], which is beneficial to the self-repair process of damaged tissues. Ca²⁺ has low cytotoxicity and is a cross-linking agent widely used in tissue engineering. Ca²⁺ can activate platelets and coagulation factor VII [39–41]. The hydrogel has a three-dimensional structure similar to the natural extracellular matrix (ECM) [42]. More importantly, it has strong plasticity and can be filled in damaged parts with complex shapes and arbitrary sizes [43]. Fibroblasts produce ECM, which plays a key structural role in the healing process of tissue damage. Hydrogel can stimulate fibroblasts to produce ECM by triggering signal pathways such as phosphoinositide 3-kinase (PI3K) and phosphorylation of protein kinase B (Akt) [44]. Therefore, Gel/SA/CMCS hydrogel has a certain procoagulant effect and is beneficial to the recovery of damaged tissues.

Carrier cells are the key to Cell-based tissue engineering's role in tissue repair. Hydrogel biomaterials must not only maintain the biological activity of carrier cells in vitro, but also the carrier cells must survive and function in the body. After implanting cell-inoculated transplants in athymic nude mice, we tracked the survival of adult stem cells in nude mice on the 7th and 14th days. The results showed that Gel/SA/CMCS hydrogel as a cell carrier can maintain the cells survive normally in vivo.

In conlcusion, this study mainly compared the survival status of two adult stem cells combined with Gel/SA/CMCS hydrogel in vivo and in vitro. The results showed no statistical difference in the growth of the two kinds of cells in vivo and in vitro, indicating that the Gel/SA/CMCS hydrogel has a high-porosity 3-dimensional structure and good tissue compatibility. Gel/SA/CMCS hydrogel will provide more options for tissue damage repair research, especially non-renewable tissues and inert tissues. Therefore, Gel/SA/CMCS hydrogel is a strong candidate for biological tissue engineering materials.

Declarations

Conflict of interest

The authors declare they have no conflicts of interest.

Ethical statement

All animal experiments were conducted in the Central Laboratory of Affiliated Hospital of Jining Medical University, according to the guidelines of the Institutional Animal Care and Use Committee and the Declaration of Helsinki.