Human mesenchymal stem cells (hMSCs) expanded with and without fibroblast growth factor (FGF) supplementation were compared with respect to their proliferation rate, ability to differentiate along the chondrogenic pathway in vitro, and their gene expression profiles. hMSCs expanded in FGF-supplemented medium were smaller and proliferated more rapidly than hMSCs expanded in control conditions. Chondrogenic cultures made with FGF-treated cells were larger and contain more proteoglycan than those made with control cells. Furthermore, aggregates of FGF-treated cells lacked the collagen type I-positive and collagen type II-negative outer layer characteristic of aggregates of control cells. A total of 358 unique transcripts were differentially expressed in FGF-treated hMSCs. Of these, 150 were upregulated and 208 downregulated. Seventeen percent of these genes affect proliferation. Known genes associated with cellular signaling functions comprised the largest percentage ( approximately 20%) of differentially expressed transcripts. Eighty percent of differentially expressed extracellular matrix-related genes were downregulated. The present findings that FGF-2 enhances proliferation and differentiation of hMSCs adds to a growing body of evidence that cytokines modulate the differentiation potential and, perhaps, the multipotentiality of adult stem cells. With the generation of gene expression profiles of FGF-treated and control cells we have taken the first steps in the elucidation of the molecular mechanism(s) behind these phenomena.

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It is well known that adult cartilage lacks the ability to repair itself; this makes articular cartilage a very attractive target for tissue engineering. The majority of articular cartilage repair models attempt to deliver or recruit reparative cells to the site of injury. A number of efforts are directed to the characterization of progenitor cells and the understanding of the mechanisms involved in their chondrogenic differentiation. Our laboratory has focused on cartilage repair using mesenchymal stem cells and studied their differentiation into cartilage. Mesenchymal stem cells are attractive candidates for cartilage repair due to their osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture. However, the need for chondrogenic differentiation is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes. This chapter will focus on the methods involved in the isolation and expansion of human mesenchymal stem cells, their differentiation along the chondrogenic lineage, and the qualitative and quantitative assessment of chondrogenic differentiation.

We have developed an improved method for preparing cell aggregates for in vitro chondrogenesis studies. This method is a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates. These modifications allow a high-throughput approach to chondrogenic cultures, which reduces both the cost and time to produce chondrogenic aggregates, with no detrimental effects on the histological and histochemical qualities of the aggregates. We prepared aggregates in both systems with human bone marrowderived mesenchymal stem cells (hMSC). The aggregates were harvested after 2 and 3 weeks in chondrogenic culture and analyzed for their ability to differentiate along the chondrogenic pathway in a defined in vitro environment. Chondrogenic differentiation was assessed biochemically by DNA and glycosaminoglycan (GAG) quantification assays and by histological and immunohistologic assessment. The chondrogenic cultures produced in the 96-well plates appear to be slightly larger in size and contain more DNA and GAG than the aggregates made in tubes. When analyzed histologically, both systems demonstrate morphological characteristics that are consistent with chondrogenic differentiation and cartilaginous extracellular matrix production.

These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/ scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.