Transient Exposure to Transforming Growth Factor Beta 3 Improves the Mechanical Properties of Mesenchymal Stem Cell–Laden Cartilage Constructs in a Density-Dependent Manner

Abstract

Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering and regenerative medicine. However, the use of these cells has been limited by their reduced ability to form functional tissue compared to chondrocytes when placed in three-dimensional culture systems. To optimize MSC functional chondrogenesis, we examined the effects of increasing seeding density and transient application of transforming growth factor beta 3 (TGF-b3), two factors previously shown to improve growth of chondrocyte-based constructs. Chondrocytes seeded in agarose at 20 million cells=mL and MSCs seeded at 20 or 60 million cells=mL agarose were cultured for 7 weeks under continuous or transient application of TGF-b3. In the transient group, cell-laden constructs were exposed to TGF-b3 for the initial 3 weeks, followed by 4 weeks of culture in medium without TGFb3. Compressive properties, biochemical content, and gene expression were assessed at 3, 5, and 7 weeks. Matrix distribution and collagen type was determined using histology and immunohistochemistry, and chondrogenic and osteogenic markers were assessed using real-time polymerase chain reaction. When maintained continuously with TGF-b3, chondrocyte-seeded constructs achieved a higher equilibrium compressive modulus than MSCs similarly maintained. Although properties of both groups increased with respect to starting values, there was no difference in bulk mechanical or biochemical properties with higher seeding density when MSCs were cultured with constant TGF-b3. Findings also showed that while transient application of TGF-b3 elicited robust growth from chondrocyte-laden gels, MSCs seeded at the same density failed to respond, although constructs maintained their previously accrued properties and continued to express cartilaginous genes after TGF-b3 removal. Conversely, MSCs seeded at 60 million cells=mL exhibited a strong anabolic response with transient TGF-b3 exposure, achieving an equilibrium modulus of approximately 200 kPa. Although this represents the highest modulus we have been able to achieve with MSC-seeded constructs using our culture system, further work remains to optimize MSC chondrogenesis for cartilage tissue engineering, particularly in terms of collagen content and dynamic mechanical properties.

Expansion in the presence of FGF-2 enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs

“…In this study the withdrawal of TGF-after 21 days did not enhance the subsequent functional development of the engineered construct, consistent with our previous findings (Buckley et al 2010b). A strategy of withdrawing TGF-from the culture media may eventually overcome the problem of diminishing matrix synthesis in long-term culture, but may depend, among other factors, on further optimization of stem cell isolation and expansion conditions (Sampat et al 2011) and the cell seeding density (Huang et al 2009). Another concern is that the long-term culture data presented here could be indicative of some inherent inability of cartilaginous constructs engineered using IFP derived stem cells to generate a fully functional tissue once implanted into a defect, or whether these results are merely a result of suboptimal in vitro culture conditions.…”

“…MSCs and chondrocytes have been shown to be phenotypically different (Segawa et al 2009), and one implication of this may be that they synthesise different amounts of key proteoglycans such as glycosaminoglycans (GAGs) and proteins such as collagens (predominantly Type II) that provide mechanical stability. Previous investigations using MSCs isolated from various tissues have shown they generate tissues with inferior mechanical properties compared to articular chondrocytes similarly maintained in 3D culture (Mauck et al 2006;Huang et al 2009;Vinardell et al 2009;Vinardell et al 2010). Although MSCs derived from intra-articular joint tissues are more phenotypically similar to chondrocytes (Segawa et al 2009), we have shown that functionally they are still inferior to chondrocytes .…”

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

“…Moreover, the encapsulated hMSCs elaborate and deposit increasing amounts of cartilage matrix with culture, which could block interactions with the peptide with time. Several previous studies showed that a transient but optimal exposure of soluble chondrogenic factors resulted in superior subsequent neocartilage formation by hMSCs (31,39). In this study, it was demonstrated that a short-term effect of synthetic cues on cellular interactions during early stages of chondrogenesis leads to improved cartilage development in the long term.…”

Cartilage Matrix Formation by Bovine Mesenchymal Stem Cells in Three-dimensional Culture Is Age-dependent

“…TGF-b3 is consistently used in tissue engineering to elicit a chondrogenic response; however, it remains possible our age-related observations are the result of changing responsiveness to TGF-b3, which was not studied. Third, we did not evaluate hypertrophic markers; however, we have previously demonstrated bovine MSCs in agarose hydrogels do not deposit appreciable amounts of mineral or collagen Type X [24]. Finally, this work was carried out in vitro, and the performance of these cells may be altered in vivo (eg, within the synovial joint).…”