Introduction
The advances in the creation of substitutes for damaged organs using mesenchymal cells as a cellular source and scaffolding for different materials provide a substantial alternative in reconstructive surgery.
Biodegradable support structures and autologous cells called matrices1 can be formed through tissue engineering. These matrices should be biocompatible, bioabsorbable, and non-immunological. Furthermore, they should support, bind, and provide cell growth while maintaining the ability to induce angiogenesis2. Certain cartilage diseases are among the most common clinical conditions that affect quality of life, making cartilage regeneration a primary objective for the bioengineering community. To address this problem, biodegradable and biocompatible polymers, such as hydroxyapatite (HA), polylactic–polyglycolic acid, and collagen, are used as scaffolds3–5. They all are appropriate niches to stimulate mesenchymal stem cells (MSCs) toward chondral or bone differentiation6–8. Some studies have suggested that adipose tissue derived from the mesoderm is rich in MSCs, which have the ability to differentiate into various cell lineages, such as adipocytes, chondrocytes, osteoblasts, and myocytes9. The use of adipose tissue-derived mesenchymal cells in the engineering of cartilage tissue has shown similarities to bone marrow-derived MSCs, for example, in the cell’s surface markers, differentiation potential and abundance in the body10. In comparison with yields of MSCs isolated from white adipose tissue, there are 300 times more stem cells, of the same weight, than those obtained from bone marrow aspirate11,12. In addition, surgical procedures for obtention of white adipose tissue, such as lipectomy of liposuction, are safer than the bone marrow obtention procedures, commonly practiced in clinical areas13. The objective of this study was to compare the in vitro efficacy of three different scaffolds for the formation of hyaline chondral tissue from MSCs obtained from visceral adipose tissue.
Methods
Isolation and culture rabbit adipose-derived mesenchymal stem cell (rADMSC)
Adipose tissue was obtained from two male New Zealand rabbits (weight, 3 kg). The animals were anesthetized with sodium pentobarbital (Pisabental, PiSA®) 45 mg/kg intravenously and xylazine (Rompun, Bayer® 1 mg/kg) and atropine (Atropisa, PiSA®) 0.05 mg/kg intramuscularly. The rabbits were placed in a dorsal decubitus position; 5 g of adipose tissue was extracted through a 2 cm infraumbilical midline incision. Ultimately, euthanasia was reached through an overdose of sodium pentobarbital (Pisabental, PiSA®) (90-120 mg/kg) intravenously.
The samples were digested for 45 min at 37°C with shaking at 200 rpm in Dulbecco’s modified eagle’s medium (DMEM) (GIBCO, Grand Island, NY) containing 0.1% type I collagenase (Worthington Biochemical, Lakewood, NJ). The cells were passed through a 70-μm filter and centrifuged at 1,200 rpm for 5 min. The cells were seeded at 50,000 cells/cm2 and maintained in DMEM supplemented with 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin/streptomycin (GIBCO). The medium was changed every 2 days until the cells reached a confluence of 80-90%. After 14 days of cell culture and expansion, the cells were removed from the culture dishes using trypsin (Gibco® cat. 25300).
Cellular differentiation
The rADMSC was used to carry out the differentiation tests toward the different mesenchymal cell lineages. These were seeded in 12-well culture plates and were stimulated by applying commercial differentiation media. For the differentiation toward the bone lineage, the MesenCultTM Osteogenic Stimulatory Kit medium (Stem Cell Technologies, United States) was used; for differentiation toward the adipose lineage, MesenCultTM Adipogenic Differentiation Medium (Stem Cell Technologies, United States) was used; and for the differentiation toward chondral lineage, MesenCultTM-ACF Chondrogenic Differentiation Medium (Stem Cell Technologies, United States) was used. To corroborate cell differentiation, Alcian Blue, Von Kossa, and Nile Red stains were performed to detect chondral, bone, and adipose lineages, respectively.
Generation of constructs by seeding rADMSCs onto HA, polylactic and polyglycolic acid (PLA-PGA), and type I collagen
rADMSCs were cultured on three different scaffolds: HA (Nukbone®, Biocriss SA Mexico), PLA-PGA (SYNTHES 2006 5/3-5/36 Laboratories, Paoli, PA), and type I collagen (Fibroquel® Aspid Pharma). Under sterile conditions, scaffold fragments (diameter, approximately 0.5 cm) were cut and placed into 12-well culture plates. Mesenchymal stem cells previously counted and pelleted were seeded onto each scaffold at a concentration of 30,000 cells/cm2 and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. The constructs were cultured for 2 weeks at 37°C and 5% CO214. The cells were cultured for 14 days, changing the culture medium every 2 days.
Adhesion and proliferation of rADMSCs
Adhesion and proliferation were evaluated through the viability of rADMSCs on the 1st, 7th, and 14th day after seeding; the analysis was conducted through fluorescence microscopy using a LIVE/DEAD viability/cytotoxicity for mammalian cell Molecular Probes kit (Thermo Fisher Scientific, Rockford, IL). Following the technical specifications established by the manufacturer, 1 μM calcein AM and 2 μM ethidium homodimer-1 were diluted in Hank’s medium. The constructs were incubated in this medium for 45 min at 37°C. In the end, the constructs were washed with PBS and analyzed using confocal microscopy (LSM 780) and ZEN 2010 Carl Zeiss software (Zeiss, Jena-Thuringia, Germany)15.
Differentiation of rADMSCs to cartilage
The characteristics of the extracellular matrix (ECM) assessed the differentiation of mesenchymal cells to chondral tissue through Alcian blue and safranin O staining at the end of the culture phase (day 14). Immunohistochemistry with antibodies directed against components of the chondral ECM: the growth factor Sox9, type II collagen (expression of type II collagen), and aggrecan (AGR) evaluated the expression of chondral tissue markers.
The constructs were stained with Alcian blue, pH 2.5, for the identification of general cell types, for assessment of the presence or absence of morphological alterations, and assessment of the synthesis of sulfated GAG-containing proteoglycans. Sox9 is expressed in all chondroprogenitor cells, predominantly in mesenchymal condensations and cartilage16. Type II collagen and AGR are expressed in ECM typical of cartilage tissue.
Ethical considerations
All animals were cared for according to the recommendations of the Care of Laboratory Animals formulated by the National Society for Medical Research, The Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, Revised 1985, US Government Printing Office, Washington, DC) and the Mexican Official Standard NOM-062-ZOO-1999. The study was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) (INP 022/2015).
Statistical analysis
Three specific scaffold types were utilized with 15 cell cultures each. Cell count was evaluated on the 1st, 7th, and 14th days. Because a normal distribution was observed, their respective mean and standard deviation (SD) were analyzed at different pre-established times.
Values were expressed as mean SD for normally distributed continuous variables, and median interquartile ranges for skewed variables.
To evaluate adhesion, an increase in the cells in the different scaffolds was analyzed and the intervention groups were compared by t-test. To evaluate proliferation, a repeated-measures analysis of variance (normally distributed) was performed considering differences between the time. As a sensibility analysis to confirm a trend during the time, p for trend was carried out.
To compare percentages of differentiation Kruskal–Wallis test (skewed) and Bonferroni post hoc test were used to compare among the different scaffolds. The analysis was performed using STATA (Version 16.1 StataCorp, College Station, TX), and p < 0.05 was established for statistical significance.
Results
rADMSC isolation and culture
Mesenchymal stem cells that showed a higher proliferation index and maintained their original phenotype were isolated during the 1st day of culture, subsequently changing their morphology to a fibroblastoid phenotype on day 14 (Fig. 1).
Figure 1. Rabbit adipose-derived mesenchymal stem cells (rADMSCs): isolation and culture. Morphological changes in rADMSCs as a function of time. The phenotypic change to fibroblastoids was observed on day 7 (×10 magnification).
Adhesion and proliferation of rADMSCs
A total of 30,000 mesenchymal cells/cm2 were seeded per culture plate. On the 1st day of culture, adhesion was quantified, ranging from 12.4 ± 2.7 × 103 to 15.8 ± 1.7 × 103 mesenchymal cells on the different scaffolds. The differences among scaffolds were significant; PLA-PGA showed the greatest number of adhered cells (Fig. 2).
The second graph depicts proliferation overtime on the three scaffolds. The collagen matrix reached the greatest proliferation significantly on the 14th day (p < 0.001 for the trend) (Fig. 3).
Figure 2. Adhesion of rabbit adipose-derived mesenchymal stem cells (rADMSCs) on the 1st day of all scaffolds. The figure depicts the adhesion of rADMSCs on the three different scaffolds; the differences among scaffolds were significant, and polylactic and polyglycolic acid showed the greatest number of cells adhered.
Figure 3. The proliferation of rabbit adipose-derived mesenchymal stem cells. The figure shows the mean ± standard deviation of the three groups on day 1, day 7, and day 14. Repeated-measures analysis of variance was performed (p < 0.05 for the 3 variables) and p for trend (< 0.001 for all).
Fig. 4 shows the adherence, survival, and proliferation of cells in different constructs over time. Cell morphology was similar throughout the different scaffolds.
Figure 4. Three-dimensional constructs were observed during the proliferation phase at different times. Comparison of scaffolds. Mesenchymal cells on different scaffolds. Visualization of the cells adhering, surviving, and proliferating on the different matrices during the 14 days of the experiment. Nuclear staining with calcein (×40 magnification).
Differentiation of rADMSCs to cartilage
Histochemical staining was performed with Alcian blue to demonstrate the presence of sulfated compounds, similar to those that make up the ECM of cartilage. Staining with safranin O revealed the production of glycosaminoglycans, which are secreted by mature chondrocytes, manifested with a nucleus that was stained red. These compounds were produced on the three scaffolds, shown in Fig. 5.
Figure 5. Characteristics of the extracellular matrix: Alcian blue and safranin O staining. Day 14 of proliferation. Alcian blue and safranin O histochemical staining. The Alcian blue and safranin O stains show the production of sulfated proteoglycans and glycosaminoglycans, respectively, on the three scaffolds; this production is similar to that in the extracellular matrix of cartilage.
The PLA-PGA scaffold showed greater differentiation toward chondral cartilage, presented with a higher percentage of cells that were positive for type II collagen and AGR. However, the type I collagen scaffold expressed a higher percentage of Sox-9-positive cells (Table 1 and Fig. 6).
Table 1. Hyaline cartilage cellular differentiation. Cell percertage (P50 min-max)
| Marker | PLA-PGA | Type I collagen | Hydroxyapatite | p-value |
|---|---|---|---|---|
| SOX9 (% cells) | 29.5 (25.5-33.3) | 42.0 (24.6-47.8)* | 29.5 (20.7-37.5) | 0.0047 |
| Aggrecan (% cells) | 40.2 (31.5-55.3)* | 33.9 (28.7-54.1) | 24.8 (22.3-33.6) | 0.0001 |
| Type II collagen (% cells) | 23.4 (18.5-44.4)* | 14.4 (5.3-24.4) | 14.8 (12.2-24.5) | 0.001 |
|
Statistic Test Kruskal–Wallis. * Post hoc test Bonferroni. PLA-PGA: polylactic and polyglycolic acid. |
||||
Figure 6. Characteristics of the extracellular chondral matrix: immunohistochemical reaction for Sox-9, type II collagen, and aggrecan (AGR) (day 14). Immunohistochemical reactions for Sox9, type II collagen, and AGR. A higher positivity to Sox9 is observed in the collagen scaffold, and the largest number of cells expressing type II collagen and AGR are found on the polylactic and polyglycolic acid scaffold (×20 magnification).
Discussion
The adhesion and proliferation of rADMSCs in the different scaffolds can be appreciated in the results of the study. They manifested a three-to-four-fold increase in the number of cells compared with the initial count, and collagen matrix had the best proliferation.
The differentiation at the end of the experiment had significant relevance according to the chondral markers expressed by the mesenchymal cell in the PLA-PGA scaffold. The differentiation is consistent with those reported by Wei et al., who used a scaffold composed of fibrin-PLGA, which improves hydrophilicity, cell adhesion, proliferation, differentiation, matrix synthesis, and cartilage regeneration17. Although the three scaffolds have been tested regarding the regeneration of different tissues in the clinical field4,18,19, the use of scaffolds with PLA-PGA to generate mature hyaline cartilage could be a better option in terms of chondral differentiation.
A large number of mesenchymal cells were obtained from rADMSCs; these cells differentiated to a chondral lineage on all scaffolds without the addition of exogenous growth or differentiation factors, as described previously20–22. It has been described that MSCs obtained from adipose tissue that has a phenotype and differentiation capacity in vitro very similar to those acquired from bone marrow but with a greater proliferation capacity. An additional advantage is that they can be obtained from surgical procedures, such as liposuction or from an abdominoplasty. Our results showed that the implantation of rADMSCs on scaffolds presents the potential for chondral cell regeneration, the basement for the treatment of cartilage defects. We believe that there are sufficient bases for its study in a clinical environment, overcoming the disadvantages related to the collection and implantation of chondrocytes23.
It has been proposed in tissue engineering that matrices must be biodegradable and designed to support cell growth to give rise to functional tissue24. The degradation of these matrices or scaffolds must correspond to the percentage of regeneration of the affected tissues. Consequently, the selection of the material is very important for the facilitation of adequate generation of tissues or organs from cells1. Based on the results of what regarding cartilage engineering, the considerations were the following: the matrix should be biocompatible, biodegradable, and replaced by the ECM produced by the hosting cells, without cytotoxic residues or inflammatory reactions. Porosity was also an important characteristic that facilitated the diffusion of growth factors and the production of ECM. The three scaffolds analyzed facilitated the processes of adhesion, proliferation, and differentiation25.
In the scaffold selection for clinical use, one should consider the physicochemical characteristics of the scaffold utilized to create a viable construct. As previously described, there are notable differences in adherence, proliferation, and differentiation according to the scaffold that was used in this in vitro study; however, they should be re-evaluated for future in vivo studies.
Conclusion
The three different scaffolds facilitated the formation of hyaline-like cartilage from adipose tissue derived from mesenchymal cells. Compared with the other scaffolds tested, the PLA-PGA scaffold showed the greatest number of adhered cells and the highest percentage of cell differentiation to the chondral lineage in vitro 14 days after seeding.
Acknowledgment
The authors would like to thank the support from Miguel Cuellar Mendoza, MD, for his technical assistance during this study and PhD Miguel Angel Jiménez-Bravo Luna‡ for his contribution.
Funding
The authors declare that this work was financially supported by the National Institute of Pediatrics through the E022 Program.
Conflicts of interest
The authors declare no conflicts of interest.
Ethical disclosures
Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
Confidentiality of data. The authors declare that no patient data appear in this article. Furthermore, they have acknowledged and followed the recommendations as per the SAGER guidelines depending on the type and nature of the study.
Right to privacy and informed consent. The authors declare that no patient data appear in this article.
Use of artificial intelligence for generating text. The authors declare that they have not used any type of generative artificial intelligence for the writing of this manuscript nor for the creation of images, graphics, tables, or their corresponding captions.
