Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study

Abstract

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes’ expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation

“…These results are in accordance with the study by Khan et al [47], where a simultaneous increase in both proliferation and differentiation of ADSCs were obtained when VEGF-A165 was combined with FGF-2. In general, our results are in line with all studies, in which FGF-2 is used for expansion of various cell types, such as mesenchymal stem cells including ADSCs [17], induced pluripotent stem cells [19] or chondrocytes [18], or for various regenerative therapies, in which the cell proliferation is needed, such as healing of cutaneous wounds [20,21], regeneration of damaged or diseased bone tissue [22,24], or treatment of vitiligo, requiring proliferation of melanocytes [26].…”

“…However, the effect of our recombinant growth factors was tested on two cell types (with different results obtained for each cell type) and in a wide range of concentrations. These concentrations were inspired by the literature, where lower concentrations (5-100 ng/mL) have been usually used for studies with soluble growth factors (e.g., [2,12,15,[17][18][19]47,49]), while higher concentrations (0.5 -10 μg/mL) have been used for functionalization of various biomaterials (e.g., [12,14,15,33,34,36,37]) to simulate a local concentration of growth factors bound to the natural ECM in vivo, which can act as reservoirs of these factors, enabling their continuous release and delivery to cells [68].…”

“…It maintains the stem cell phenotype of ADSCs, enhances their proliferation, and thus it is suitable for ADSC expansion and maintenance of their therapeutic potential [17]. In addition, it is a component of media for the expansion of various cell types, e.g., chondrocytes [18] or induced pluripotent stem cells [19]. FGF-2 has been widely applied in clinical practice, particularly in cutaneous wound healing, such as second-degree burns and chronic ulcers [20,21], and in bone regenerative therapies, such as treatment of bone fractures, osteotomies, and osteonecrosis (for a review, see [22]), or treatment of periodontitis with intrabony defects ( [23]; for a review, see [24]).…”

Chondrocyte De-Differentiation: Biophysical Cues to Nuclear Alterations

“…However, large quantities of chondrocytes are needed for cartilage repair, which makes ex-vivo expansion essential for any potential use in ACI. When chondrocytes are expanded, they are cultured in a monolayer 2D culturing system, which is an effective method that generates a large number of viable cells [ 45 ]. Several commercial ACI expansion companies use two-dimensional monolayer systems to provide patients with 4 to 12 million viable cells for implantation ( Table 1 ).…”

The Feasibility of Using Human Primary Chondrocytes Derived from Osteoarthritic Patients Overexpressed with SOX9 Seeded on PLGA-Fibrin Hybrid Scaffolds for Cartilage Engineering

“…Multiple studies using nasal septum [65][66][67] and ear [68,69] cartilages showed that the constructs formed by these two cells sources survived subcutaneously even after six weeks [69] and six months [65] of implantation in nude mice. Unlike nasal septum and ear cartilages, articular cartilage lines the epiphyses of joint ends of the bone.…”