Enhancement of chondrogenesis of human adipose derived stem cells in a hyaluronan-enriched microenvironment
Introduction
Articular cartilage has limited capacity to repair damage caused by trauma or disease because of its avascularity and low cellular mitotic activity [1]. The chondral lesions often result in progressive deterioration and eventual osteoarthritis [2]. Although total joint arthroplasty is one of the most common indications of a diffuse lesion, therapies for focal defect such as microfracture, multiple drilling, and cartilage gouging are also performed. However, current strategies are not able to restore the native structure of cartilage and may even increase the risk of further damage [3], [4]. Accordingly, cell-based articular cartilage tissue engineering is a new emerging method that offers advantages over current treatment strategies [5].
In recent years, cell-based articular cartilage tissue engineering studies were focused on using either differentiated chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs) for transplantation [6]. However, limited proliferative capacity of differentiated chondrocytes and BMSCs possess a major challenge in providing adequate cell numbers for viable transplantations and cartilage repair. More importantly, the ex vivo expansion of chondrocytes results in a loss of their phenotypes [7], and the proliferative capacity of BMSCs are age dependent [8], [9]. Adipose derived stem cells (ADSCs) have the following advantages over the aforementioned methods: (i) can be obtained with relatively little discomfort, (ii) causes lower donor site morbidity, and (iii) can be expanded to large numbers in vitro [10], [11]. Therefore, ADSCs may be a more feasible choice as a cell source for cell-based tissue engineering for cartilage regeneration.
Ideal cartilage scaffolds would play a pivotal role in dictating cell adhesion, proliferation, and/or differentiation for expressing desirable phenotypes to regenerate cartilage. Poly-lactic-co-glycolic acid (PLGA) is a safe biomaterial for clinical applications, and has been approved by the Food and Drug Administration (FDA), U.S.A [12], [13]. Structural modifications to PLGA have yielded fine fibrillar meshworks and foams, which have been exploited during the past 10 years for tissue engineering purposes [14]. However, PLGA, as a synthetic polymer, can offer better control of the matrix architecture and chemical composition, but has relatively low biological activity. Reports have indicated that the attachment of specific bioactive elements (such as proteins or peptides) to the polymer scaffold can regulate activities of seeded cells [15], [16], [17], [18]. On the other hand, the extracellular matrices (ECMs) provide a microenvironment for cells to maintain homeostasis and differentiation properties for specific tissues [1], [18], [19], [20], [21]. Among the ECMs, hyaluronan (HA) is the main glycosaminoglycan in the mesenchyme during the early stage of chondrogenesis [1], [22], [23], [24], [25], [26], [27]. Most importantly, HA is the major physiological component of the articular cartilage matrix, and is particularly abundant in synovial fluid. Accordingly, we hypothesized that immobilizing HA on surface of the biomaterial may provide a suitable microenvironment for hADSCs to differentiate into the chondrogenic lineage, and thus produce a cartilage-specific matrix for articular cartilage regeneration. To test the hypothesis, we examined the chondrogenesis of hADSCs by culturing the cells in HA-coated wells and HA-modified PLGA (HA/PLGA) scaffolds.
Section snippets
Materials
Two series of PLGA [lactide:glycolide ratio 50:50 (50/50) and 75:25 (75/25)] were purchased from Sigma–Aldrich (St. Louis, MO, USA). The polyethyleneimine (PEI) (Mw = 423) was purchased from Aldrich. Sodium hyaluronate, grade FCH-200 (Mw = 2–2.1 MDa), was obtained from Kibun Food Chemicals (Tokyo, Japan). N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum
Chondrogenesis of hADSCs in HA-enriched microenvironment
The effect of the HA-enriched microenvironment on chondrogenesis of hADSCs was determined by cell aggregation, the mRNA expressions of marker genes in chondrogenesis and synthesis of cartilage-specific matrix were evaluated by culturing hADSCs in different amounts of HA-coated wells (0.005, 0.025, 0.05, and 0.5 mg/cm2 per well). In comparison to the control group (without HA coating), higher cell aggregation was found in HA-coated groups (0.005∼0.5 mg/cm2) (Fig. 1) 24 h after plating, and the mRNA
Discussion
Articular hyaline cartilage injuries still pose a big challenge to orthopedic surgeons, because these defects have poor capacity for intrinsic repair. Construction of a 3D biomaterial with autologous adult stem cells to regenerate defected articular cartilage may become a viable clinical option. Previous results have indicated that PLGA scaffolds are useful matrices for allowing controlled scaffold degradation in vivo, but they demonstrate low biological activity. On the other hand, HA
Conclusion
We found that the HA-coated well and HA/PLGA scaffold mimicking the HA-enriched ECM provides a suitable microenvironment to enhance chondrogenesis in hADSCs. The gene expression profile and ECM formation were evidence that the HA/PLGA scaffold leads hADSCs toward chondrogenesis and to synthesize hyaline cartilage, but not fibrous cartilage. Our results emphasize that a HA-enriched microenvironment may be applied for more effective articular cartilage tissue engineering.
Acknowledgement
This study was supported by grants from the National Science Council (NSC 97-2321-B-037-001-MY2) in Taiwan, ROC.
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