Biomaterials for cartilage tissue engineering should provide biochemical stimuli and a structural environment, which are important for inducing chondrogenesis and retaining the phenotypes of differentiated stem cells.23
Many synthetic or naturally derived polymers have been extensively studied in cartilage tissue engineering, such as biodegradable polyesters (e.g., poly(lactic-co-glycolic acid)), polyurethane, poly(3-hydroxybutyrate), hyaluronic acid, collagen, fibrin and gelatin.24–28
Recently, decellularized tissues have gained increasing attention for their excellent biocompatibility and bio-inductive properties.29–37
ECM containing endogenous bioactive factors, derived from a variety of intact tissues, has been applied as an allograft or xenograft material for tissue engineering, including for adipose,19
Indeed, ECM secreted from the resident cells of tissue is an ideal biomaterial evolved by nature. ECM plays numerous critical roles in the lives of cells. ECM provides mechanical support for cells and tissues, integrate cells into tissues, influences cell shape and movement, influences mechanical and chemical signaling pathways, and coordinates the behavior of different cells in tissues.9
We successfully fabricated a composite of hECM and stem cells containing various bioactive factors. An important consideration in tissue engineering scaffold design is the provision of a framework for cellular events. The hECM scaffold exhibited viscous gel-like appearances and a fibrous microstructure (). From a structural viewpoint for scaffolds, a fibrous and gel-like structure is highly desirable. A porous and fibrous structure provides surfaces for cell attachment and initial biomechanical stability.41,42
In particular, hydrogels mimic the microenvironment found in cartilage.43,44
More importantly, the hECM scaffold contained bioactive factors such as TGF-β1, IGF-1, bFGF, and VEGF, which could act as major biochemical cues for chondrogenesis (). These growth factors are known to induce chondrogenic differentiation of stem cells and to aid in the maintenance of phenotype.45,46
In particular, major cartilage modulating growth factors are TGF-β1 and IGF-1. TGF-β1 is a potent factor in proliferation and differentiation of stem cells into chondrocytes. IGF-1 is synthesized by chondrocytes and prevents chondrocyte apoptosis. In addition, both TGF-β1 and IGF-1 is well known for the ability to stimulate production of cartilage matrix, including collagen, proteoglycan, and hyaluronan (HA).46,47
Therefore, the structural and biochemical properties of the hECM scaffolds could help enhance the adhesion, proliferation, and chondrogenic differentiation of stem cells. High-cell density hASC/hECM composites may be favorable for promoting the cell-matrix and cell-cell contacts.
Biomaterials in combination with autologous adult stem cells have been studied to regenerate defected cartilage.1
Multipotent mesenchymal stem cells are attractive because of their self-renewing potential and multipotentiality. In particular, hASCs obtained from autologous adipose tissue are useful because adipose tissue is abundant, readily accessible, and easily expandable.48
hASCs can be induced toward a chondrogenic phenotype by exogenous growth factors, including TGF-β1, IGF-1, and BMP-4.15,16
When hASCs were cultured in combination with hECM scaffolds for 45 days, the composites formed spherical cartilage-like tissue. The composite size and weight gradually increased with culture time (). SEM showed that the hASCs were well-attached to the fibrous ECM and formed dense aggregation after 45 days, and a live/dead assay also indicates that most cells in the composite remained viable ( and ). These results suggest that the hECM scaffold provides a good microenvironment favorable to the adhesion of cells and the maintenance of viability. Moreover, the rapid increase in DNA content also indicated that the scaffold well supported the growth of hASCs (). The synthesis of cartilage-specific proteins, including collagen and sGAG, as a key marker of chondrogenesis, substantially increased with time when the hASCs were cultured with the hECM scaffold in chondrogenic medium (). Although all of the cartilage-related proteins were expressed in higher levels when TGF-β1 was added, the chondrogenesis of hASCs was successfully induced without additional TGF-β1. Therefore, our results imply that the hECM gel-like scaffold could promote the synthesis and deposition of cartilage-specific proteins in vitro
, highlighting the importance of hECM scaffolds in the differentiation of hASCs toward chondrogenesis.
Chondrogenesis involves the formation of cartilage tissue through stem cell differentiation and is tightly controlled by regulation of gene expression and cell-cell or cell-ECM interactions. Condensation and proliferation of stem cells is one of the earliest events in chondrogenesis, and condensed stem cells begin up-regulating the transcription factor Sox-9
and producing cartilage-specific ECM proteins including AGN
, proteoglycans, HA, and collagen type II, VI, IX, XI.49,50
Collagen type X is expressed later, when chondrocytes acquire the hypertrophic phenotype, form calcified cartilage, or enter endochondral ossification. There is a concern on terminal differentiation into hypertrophic chondrocytes, which may lead to inter-regional osteophytes during in vivo
In this study, the expressions of chondrogenic markers including Sox-9
, and collagen type II, XI were promoted in hASC/hECM composites, as were increase in sGAG and collagen contents. In contrast, collagen X mRNA was slightly expressed only in hASC/hECM composites with TGF-β1 on day 45 (). These results confirm that the hECM scaffold derived from human adipose tissue was able to trigger and support chondrogenic differentiation of hASCs.
FIG. 9. Reverse-transcription-polymerase chain reaction analysis of gene expression for chondrogenic differentiation in hASC/hECM composites. hASC undifferentiated cells (A) were used as a negative control (lane 2). Each expressed gene was analyzed using human-origin (more ...)