Elsevier

Biomaterials

Volume 31, Issue 4, February 2010, Pages 631-640
Biomaterials

Enhancement of chondrogenesis of human adipose derived stem cells in a hyaluronan-enriched microenvironment

https://doi.org/10.1016/j.biomaterials.2009.09.089Get rights and content

Abstract

Microenvironment plays a critical role in guiding stem cell differentiation. We investigated the enhancing effect of a hyaluronan (HA)-enriched microenvironment on human adipose derived stem cell (hADSC) chondrogenesis for articular cartilage tissue engineering. The hADSCs were obtained from patients undergoing hip replacement. HA-coated wells and HA-modified poly-(lactic-co-glycolic acid) (HA/PLGA) scaffolds were used as the HA-enriched microenvironment. The mRNA expressions of chondrogenic (SOX-9, aggrecan and collagen type II), fibrocartilage (collagen type I), and hypertrophic (collagen type X) marker genes were quantified by real-time polymerase chain reaction. Sulfated glycosaminoglycan (sGAG) deposition was detected by Alcian blue, safranin-O staining, and dimethylmethylene blue (DMMB) assays. Localized collagen type II was detected by immunohistochemistry. The hADSCs cultured in HA-coated wells (0.005–0.5 mg/cm2) showed enhanced aggregation and mRNA expressions (SOX-9, collagen type II, and aggrecan) after 24 h, and sGAG content was also significantly increased after 9 days of culture. The HA-modified PLGA did not change the cell adherence and viability of hADSCs. The mRNA expressions of chondrogenic marker genes were significantly enhanced in hADSCs cultured in HA/PLGA rather than those cultured in the PLGA scaffold after 1, 3, and 5 days of culture. The hADSCs cultured in HA/PLGA produced higher levels of sGAG and collagen type II, compared to those in the PLGA scaffold after 4 weeks of cultures. Our results suggest that HA-enriched microenvironment induces chondrogenesis in hADSCs, which may be beneficial in articular cartilage tissue engineering.

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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