Serum‐free, chemically defined medium with TGF‐β₃ enhances functional properties of nucleus pulposus cell‐laden carboxymethylcellulose hydrogel constructs

Abstract

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-beta(3)). Glycosaminoglycan (GAG) content increased in both TGF-beta(3)-treated groups and was highest for treated, serum-free constructs (9.46 +/- 1.51 microg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF-beta(3) (18.54 +/- 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 +/- 0.46 vs. 19.94 +/- 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-beta(3) supplemented medium, indicating the importance of medium formulation in NP construct development.

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review

“…Excluded studies (10,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) and reasons are described in Table 4. Most of the included studies described protocols relying on the platelets lyse after freeze-thaw cycles to obtain VBDs.…”

Functional nucleus pulposus-like matrix assembly by human mesenchymal stromal cells is directed by macromer concentration in photocrosslinked carboxymethylcellulose hydrogels

“…CDM comprises High Glucose DMEM with 1 % insulintransferrin-selenium + universal culture supplement (BD Biosciences, San Jose, CA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, 40 μg/mL L-proline (Sigma), 1 mM sodium pyruvate (Mediatech, Manassa, VA, USA), 50 μg/mL ascorbic acid 2-phosphate (Sigma) and 100 nM dexamethasone (Sigma) (Mackay et al 1998). The TGF-β3 concentration utilized was chosen based on previous IVD and cartilage tissue engineering studies (Mackay et al 1998;Risbud et al 2004;Reza and Nicoll 2010b;Gupta et al 2011). Medium was changed 3 times per week for 6 weeks and hydrogels were isolated for various forms of analysis at 1, 7, 21 and 42 days.…”

“…Following lyophilization, total protein and DNA were extracted on days 7, 21 and 42 (n=4-5) by pepsin digestion based on previous studies (Chou and Nicoll 2009;Reza and Nicoll 2010b). Briefly, lyophilized samples were homogenized and treated with pepsin (Sigma) in 0.05 N acetic acid (4,560 Units/ mL) for 48 h at 4°C.…”

“…The DMMB dye was reduced to pH 1.5 to minimize the formation of CMC carboxyl group-DMMB dye complexes (Enobakhare et al 1996) and absorbance was determined at 595 nm using a chondroitin-6 sulfate standard curve from shark cartilage (Sigma) (Reza and Nicoll 2010b;Gupta et al 2011).…”

“…Alizarin Red S (1 %) staining was used to detect calcium ion deposition in all macromer concentration groups at day 42 (Kiernan 1999) using β-tricalcium phosphate (β-TCP) scaffolds (Stryker Ortho Biologics, Malvern, PA, USA) as positive controls. In addition, immunohistochemical analyses were performed on days 7, 21 and 42 constructs according to previous studies (Chou and Nicoll 2009;Reza and Nicoll 2010b). Briefly, slides were incubated with monoclonal antibodies to COL II, COL X, type VI collagen (COL VI) (II-II6B3, X-AC9 and 5C6, respectively; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) and non-immune IgG controls (Sigma) at a concentration of 1 μg/ml in blocking solution composed of 10 % horse serum diluted in DPBS.…”