Background
The repair of cartilage defects caused by trauma, tumour and congenital factors remains a major challenge for plastic and orthopaedic surgeons because cartilage has a low spontaneous repair and regeneration capacity. Several strategies have been developed to restore and repair cartilage defects, such as microfracture, abrasion chondroplasty, cartilage scraping, and transplantation of the perichondrium, periosteum, and cartilage [
1]. However, these strategies generate tissue that cannot substitute for native cartilage, and the results of currently available therapies are far from satisfactory [
2].
Autologous chondrocyte implantation (ACI) presents encouraging results [
3]. Kuroda et al. demonstrated that a three-dimensionally structured autologous chondrocyte implant was effective in repairing cartilage defects in a rat model of anterior cruciate ligament-induced osteoarthritis (OA) [
4]. Over the past 20 years, combining biomaterial scaffolds and cell sources to induce cartilage regeneration has emerged as a promising new strategy [
5]. Fulco et al. isolated chondrocytes from the nasal septum and engineered autologous nasal cartilage tissues to repair cartilage defects after skin cancer excision [
6]. The cutaneous sensitivity and structural stability of the reconstructed area were clinically satisfactory, with adequate respiratory function [
6]. However, cell-based therapy, including chondrocyte expansion in vitro, results in loss of the chondrocyte phenotype and ageing [
7].
Owing to its multipotent differentiation potential and high proliferative potential, bone mesenchymal stem cell (BMSC)-based cartilage tissue engineering and cartilage regenerative medicine offer a promising strategy for treating cartilage defects [
8]. Jia et al. demonstrated that differentiated BMSCs combined with an oriented scaffold can successfully repair full-thickness articular cartilage defects in rabbits and produce cartilage with enhanced biomechanical properties [
9]. However, an increasing number of studies have demonstrated that although chondrogenically differentiated BMSCs show chondrogenic potential in advance, these cells tend to eventually vascularize or ossify, undergo terminal chondrocyte differentiation and are replaced by osseous tissue, which indicates that the chondrogenic differentiation of BMSCs represents a transient state only [
10].
Recently, chondrogenic stem/progenitor stem cells derived from cartilage tissue were isolated and identified. In our previous study, we isolated cartilage stem/progenitor stem cells in a pig model by a differential adhesion assay to fibronectin, and evaluated the stemness of the cartilage stem/progenitor stem cells [
11,
12]. However, there is only few research concerning the chondrogenic characteristics of cartilage progenitor cells (CPCs) in vivo [
13]. Several studies have used PHB and PHBV as biomaterials for cartilage tissue engineering [
14‐
16], and we used PHBV as a scaffold for cartilage tissue engineering and showed that PHBV scaffolds have the potential to be used as chondrocyte carriers for cartilage engineering in our previous study [
17]. In the current study, we explored the feasibility of combining CPCs with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the in vitro proliferation ability and in vivo chondrogenic characteristics of CPCs with those of BMSCs and chondrocytes.
Materials and methods
All experimental protocols involving animal tissues and cells were approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine.
Chondrocytes and CPCs were harvested from swine articular cartilage tissue via differential adhesion to fibronectin in vitro, as described previously [
18,
19]. The obtained articular cartilage tissues were minced into 1 mm
2 pieces and then washed in sterile PBS and chloromycetin thrice. The cartilage tissue was digested in high-glucose DMEM containing 0.1% collagenase type 2 in a 37 °C shaking water bath for 6–8 h. Then, the suspension was filtered through a 200-μm filter to remove undigested particles, and chondrocytes at a density of 4000 cells/ml were seeded onto 10-cm plastic dishes (treated with 10 μg/ml fibronectin overnight) at 37 °C for 20 min in low-glucose DMEM. After 20 min, non-adherent cells and media were removed, and low-glucose DMEM containing 10% FBS was added to the plates. The adherent cells were cultured for 7–14 days until the cells reached 80–90% confluence. The cells were then digested with 0.25% trypsin plus 0.02% EDTA (Invitrogen) and sub-cultured into new dishes at a density of 2 × 10
4 cells/cm
2.
Swine bone marrow was obtained from the posterior superior iliac crest of newborn pigs as described previously [
20]. Low-glucose DMEM supplemented with 10% FBS was added to the aspirate (1:1) and loaded over Percoll (Sigma, St. Louis, Mo., USA) for density gradient centrifugation. Mononucleated cells were harvested from the interface after centrifugation at 3000 rpm for 10 min and then washed twice with PBS. Cells were re-suspended in low-glucose DMEM containing 10% FBS, plated into 100-mm culture dishes at a density of 2 × 10
5 cells/cm
2 and incubated at 37 °C in an atmosphere of 5% CO
2 in air. Non-adherent cells were removed by a medium change after 24 h. Adherent cells were cultured for 7–14 days until cells reached at least 80–90% confluence. The cells were then digested with 0.25% trypsin plus 0.02% EDTA and sub-cultured in a 100-mm culture dish at a density of 2.5 × 10
4 cells/cm
2. The medium was changed twice a week until the cells were 80–90% confluent.
Cell proliferation in vitro
The cell proliferation rate was assessed with a cell counting kit (CCK)-based colorimetric assay (CCK-8; Dojindo China Co., Ltd.). Chondrocytes, BMSCs, and CPCs re-suspended in 100 μl of DMEM containing 10% FBS were seeded at a density of 1000 cells/well in 96-well plates and cultured for 1 day, 3 days, 5 days, and 7 days. Before every test, 10 μl of CCK-8 solution was added to each well and incubated for 4 h. Then, the absorbance of the supernatant was measured spectrophotometrically at 450 nm, and the test was performed in triplicate.
Preparation of PHBV scaffolds and cell-scaffold constructs
PHBV scaffolds were prepared using a solvent casting-particulate leaching method as described previously [
17]. Scaffolds cut into the shape of cylinder with the same size (5 mm diameter, 2 mm thick) were used in the study. The PHBV scaffolds were first evaluated by optical microscopy and then examined with a scanning electron microscope (SEM; EPMA-8705QH2, Shimadzu, Japan) after being coated with gold as described previously [
17], and the SEM examination was performed at an accelerating voltage of 20 kV.
Chondrocytes, BMSCs, and CPCs at passage 2 (2.5 × 106 in 40 µl) were then evenly dropped onto each scaffold. After 4–6 h of incubation to allow adequate adhesion of the cells to the scaffold, low-glucose DMEM containing 10% FBS was added to immerse the cell-scaffold construct. The constructs were then cultured in an incubator at 37 °C with 5% CO2 and 95% humidity. After 1 week of in vitro culture, the constructs were implanted subcutaneously into nude mice and harvested at 6 weeks post implantation.
Wet weight and volume measurement
The weight and volume measurement of in vivo engineered tissue were measured 6 weeks after implantation. The wet weight of each specimen was measured using an electronic balance, and the diameter and thickness of each specimen were measured with vernier callipers.
Glycosaminoglycan (GAG) and total collagen
Six weeks after implantation, the glycosaminoglycan (GAG) content of the specimens was assayed by Alcian blue colorimetric analysis as previously described [
21]: the specimens were ground to obtain a protein solution. A series of reagents was added step by step to ensure specific binding of Alcian blue to polysulfated GAG molecules in cartilage. All GAGs were precipitated specifically in guanidine-HCl using a low pH in combination with detergent and a high salt concentration. The precipitate was then dissolved in a mixture of guanidine-HCl and propanol. For quantification, absorbance was recorded using a microplate reader with a 600-nm filter, and a linear standard curve between 0.5 and 20 mg was generated by adding known amounts of proteoglycans.
The total collagen content was analysed according to previously described methods [
22]. Six weeks after in vivo transplantation, cell-scaffold constructs were rinsed with PBS and then lyophilized. Subsequently, the dry mass of lyophilized samples was measured and then hydrolysed in 6 N HCl, and the hydroxyproline concentration was analysed to determine collagen content.
Histology and immunohistochemistry
The samples harvested 6 weeks after implantation were prepared for histological and immunohistochemical examination to evaluate chondrogenesis. The specimens were first fixed in buffered 10% formalin in PBS for 4–6 h, embedded in paraffin and then cut into 5-μm sections. The sections were stained with haematoxylin and eosin (HE), safranine-O and type II collagen (COL II) to evaluate the histological structure and cartilage matrix deposition in engineered tissue. COL II expression was detected using a mouse anti-human COL II monoclonal antibody (1: 100 in PBS; Santa Cruz, Santa Cruz, Calif., USA) and a horseradish peroxidase-conjugated anti-mouse secondary antibody (1: 200 in PBS; Santa Cruz) followed by colour development with diaminobenzidine tetrahydrochloride (Santa Cruz).
GAG, total collagen and biomechanical analysis
A biomechanical analyser (Instron, Canton, Mass., USA) was used for biomechanical tests. As previously described [
23], a constant compressive strain rate of 1 mm/min was applied until a maximal force of 100 N was achieved; thus, a force–displacement curve was obtained. The compressive modulus of the tested tissue was calculated from the force–displacement curve.
Real-time quantitative polymerase chain reaction
The samples were harvested 6 weeks after in vivo implantation, total RNA was extracted from each specimen, and cDNA was obtained by reverse transcription (RT) according to previously described methods [
24], the gene expression was evaluated by real-time quantitative PCR analysis with the brilliant SYBR green qPCR kit (Stratagene, USA). The PCR reactions were performed using a real-time PCR detection system (Bio-Rad Laboratories) and thermo cycler conditions following suggestions of the manufacturer. The relative gene expression levels were determined using the 2ΔΔCT method. Aggrecan, collagen II, and sox-9, as well as VEGF, were used to evaluate chondrogenic differentiation. The primers used in this study are shown in Table
1. The β-actin mRNA level was quantified as an internal control. The experiments were repeated at least three times.
Table 1
Primer sequences for PCR
Aggrecan | NM_001135 | Sense 5′-GGGGAATCTTCTGGCATTAA-3′ | 381 |
Antisense 5′-CGTTGGAGCCTGGGTT-3′ |
SOX-9 | NM_000346.4 | Sense 5′-GGCTCGGACACAGAGAACAC-3′ | 195 |
Antisense 5′-GTGCGGCTTATTCTTGCTCG-3′ |
COL II a1 | NM_001844.5 | Sense 5′-TGCTGCTGACGCTGCTC-3′ | 294 |
Antisense 5′-GTTCTCCTTTCCTGTCCCTTTG-3′ |
VEGF | NM_001025366.2 | Sense 5′-CATCTTCAAGCCGTCCTGTGT-3′ | 142 |
Antisense 5′-TCCTATGTGCTGGCCTTGGT-3′ |
β-Actin | NM_001101.5 | Sense 5′-ACATCAAGGAGAAGCTCTGCTACG-3′ | 366 |
Antisense 5′-GAGGGGCGATGATCTTGATCTTCA-3′ |
Enzyme-linked immunosorbent assay
The VEGF content in three groups 6 weeks after in vivo implantation was quantified using ELISA kits (R&D Systems) according to the manufacturer’s instructions as previously described [
25], and the plates were incubated with 100 μl of VEGF standards and diluted samples. The intensities were determined at 450 nm using a microplate reader (Thermo Scientific, USA). The test was performed in triplicate.
Statistical analysis
Statistical evaluations were performed using an ANOVA followed by post hoc analysis. A p value less than 0.05 was considered statistically significant.
Discussion
Due to the poor self-repair and regeneration capacity of cartilage, the treatment of cartilage defects is a knotty problem, and satisfactory therapeutic options are very scarce [
26]. Advances in biomedicine and biomaterials have promoted the development of new cartilage repair techniques, and cartilage tissue engineering provides a novel alternative therapeutic option for the regeneration of cartilage tissue that is damaged due to trauma or disease [
27].
The basic approach of cartilage tissue engineering involves the application of cells, scaffolds, and a specific microenvironment alone or in combination [
28]. As a key element in cartilage tissue engineering, seeding cells play a vital role [
29]. The major challenges in cartilage engineering include selection of the seeding cell source, in vitro expansion, and differentiation [
30,
31]. Chondrocytes, BMSCs, and adipose-derived stem cells (ADSCs), as well as other cells have all been explored for their potential as an ideal cell source for cartilage regeneration [
27,
28]. Chondrocytes, the predominant cell type in cartilage, synthesize matrix components and were the first seeding cells used in cartilage tissue engineering because chondrocytes are the only cell found in native cartilage, while the poor proliferation ability and the dedifferentiation of chondrocytes are bottlenecks in the clinical application of chondrocytes [
32]. Recently, there has been increasing interest in stem cell-based cartilage tissue engineering options in surgical practice to deal with lost or damaged cartilage tissue, and BMSCs could be promising cell sources for use in cartilage regeneration [
21]. BMSCs have multipotent differentiation potential and high proliferation potential, and an increasing number of studies have demonstrated that chondrogenically differentiated BMSCs underwent vascularization or endochondral ossification after in vivo transplantation [
10,
20].
Cartilage progenitor cells, which are characterized by stem cell markers, multi-lineage ability, and their self-renewal potential, have recently been found in different cartilage tissues, such as auricular cartilage, articular cartilage, and nasal cartilage, and these stem/progenitor cells are thought to respond to injury and migrate into cartilage defect zones [
33,
34]. Therefore, in the current study, we explored the feasibility of combining CPCs with PHBV to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics CPCs with those of BMSCs and chondrocytes.
Cartilage tissue engineering requires a considerable number of seeding cells, and the proliferative ability of seeding cells plays a vital role in cartilage tissue engineering [
35]. It has been reported that mature chondrocytes that have reached the end of the differentiation process do not have the capacity to proliferate or differentiate [
36]. Chondrocytes are currently considered terminally differentiated cells and thus represent the last stage of differentiation in the chondrogenic cell lineage, and terminally differentiated chondrocytes have a limited proliferative capacity [
37]. In addition, in vitro expansion of chondrocytes leads to dedifferentiation of mature chondrocytes, which is characterized by increased expression of type I collagen, decreased expression of COL II and decreased proteoglycan content. In contrast, BMSCs have been reported to be an undifferentiated population capable of endless self-renewal and have high proliferative potential [
38]. In the current study, we first compared the proliferation characteristics of chondrocytes, BMSCs, and CPCs. The current results indicated that CPCs have a higher proliferation rate than chondrocytes and a lower proliferation rate than BMSCs.
Chondrogenic differentiation potential is an important index of stem cells [
39]. Many studies have presented the advantages and feasibility of using BMSCs to treat cartilage defects [
40]. For chondrogenic differentiation of BMSCs, the incomplete chondrogenesis and the formation of fibrocartilage remain difficult problems [
41]. During the process of chondrogenesis, BMSCs adopt a transient chondrocyte phenotype rather than a permanent state and tend to undergo terminal differentiation, which is followed by endochondral ossification [
10]. In our previous study, we also found that some chondrogenic-induced human BMSCs in vitro became ossified after implantation in vitro [
41].
The key problem of neo-cartilage tissue regenerated by mesenchymal stem cells is the failure to maintain the chondrocyte phenotype: on the one hand, the cell origin may determine the ultimate fate of the mesenchymal stem cell (MSC) regenerated cartilage tissue [
10]; on the other hand, MSCs tend to lose their chondrogenic properties simultaneously upon persistent exposure to the chondrogenic stimuli, such as TGF- β and dexamethasone, prevalent in current culture methods, indicating that exogenous factors may lead to loss of the chondrogenic phenotype and may promote chondrocyte hypertrophy and endochondral ossification [
42]. To prevent exogenous chondrogenic stimuli from affecting the chondrogenic differentiation potential, we combined the chondrocytes, BMSCs, and CPCs with the biomaterial-PHBV separately without chondrogenic induction and implanted the combination subcutaneously, and we found that the tissue formed by BMSCs without induction became obviously vascularized 6 weeks after implantation, while the cartilage engineered by chondrocytes without induction exhibited a mature cartilage appearance 6 weeks after implantation. These findings were further supported by the histology and immunohistochemistry results. More interestingly, we found that the tissue formed by CPCs without induction formed cartilage-like tissues with an ivory-whitish appearance, indicating that CPCs could differentiate into chondrocytes spontaneously without chondrogenic induction. Normal chondrocytes express high levels of COL II and aggrecan, and stem or progenitor cells express chondrocyte-specific genes only in the presence of specific induction conditions (TGF-β1 (10 ng/ml), FGF (25 ng/ml), ITS (1 : 100), and dexamethasone (10–7 M), without FBS) [
43,
44]. We also found that cartilage-derived stem cells express chondrocyte-specific genes without specific induction medium containing TGF-β1 and dexamethasone. There are two possible explanations for the spontaneous chondrogenesis of CPCs: on the one hand, BMSCs have been reported to be multipotent progenitor cells because of their capability to differentiate into several mesenchymal cells, including osteoblasts, adipocytes, tenocytes, fibroblasts, and myoblasts, rather than just chondrocytes. On the one hand, the differentiation potential of CPCs, may be mainly confined to chondrogenesis. On the other hand, CPCs are isolated from chondrocytes by a differential adhesion assay to fibronectin, and the so-called “CPCs” may be a mixture of progenitor cells and chondrocytes, not pure CPCs. CPCs may undergo spontaneous chondrogenic differentiation without chondrogenic induction, while BMSCs cannot reach a chondrogenic differentiation stage without chondrogenic induction.
Authors’ contributions
KX, XDZ, and KL conceived the idea, designed the experiments, provided their funds for the study, and revised the manuscript. KX and LQ designed the study and performed the research, data analysis, and manuscript writing. ZXG and WYX contributed to the analyses and interpretation of data. All authors read and approved the final manuscript.