A Polyethylene Glycol‐Crosslinked Serum Albumin/Hyaluronan Hydrogel for the Cultivation of Chondrogenic Cell Types

Abstract

Hydrogels are gaining increasing interest as a structural basis for regenerative cell implants. Here we present in vitro data on growth and differentiation of human mesenchymal stromal precursor cells (MSCs), adult human knee chondrocytes, and adult human intervertebral disk cells in a gel based on PEG‐crosslinked serum albumin, supplemented with 4% hyaluronan (HSA–HA). Each gel culture was compared to its parallel proliferating monolayer (PML) culture and non‐proliferating high density monolayer culture (HDML). MSCs were chondrogenically induced in both standard micromass (MM) cultures and HSA–HA. Cell viability in HSA–HA was above 90%. Chondrogenic differentiation was defined by changes in the expression of the mRNA for collagen types I, II, and X, for aggrecan, for alkaline phosphatase (ALP), and for hyaluronan synthases‐2 and ‐3 (has‐2, has‐3). For disk cells and chondrocytes, the changes for collagen type II and aggrecan were in the range of up to 100‐fold, depending on the human individual. After induction, MSCs differentiated equally well in MM and HSA–HA, showing increases spanning several orders of magnitude compared to uninduced monolayer (ML) cells. For has‐2 and has‐3, the changes were less obvious, but interestingly, the ratio of has‐2/has‐3 expression in HSA–HA cultures decreased approximately 40‐fold for disk cells, remained unchanged for chondrocytes, but increased sevenfold for MSCs, when compared to the corresponding ML cultures. Taken together, HSA–HA revealed its potential as an anchoring hydrogel for chondrogenic cell types.

Maintenance of “stem cell” features of cartilage cell sub-populations during in vitro propagation

“…Gene expression was analyzed by semi-quantitative real-time PCR using an Applied Biosystems 7500 Fast Real-Time PCR System as described in [34]. The qPCR mastermix plus for SYBR green I (low ROX) kit from Eurogentec was used.…”

“…HSA-HA preparation and cell embedding in hydrogels are described in [34]. The gels were overlaid with either 1.5 ml chondrocyte culture medium (CM) or chondrogenic induction medium (DMEM high glucose, 4.5 g/l (Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM L-glutamine, 1 mM sodium-pyruvate, 170 μM ascorbic acid 2-phosphate, 0.1 μM dexamethasone, 1 × ITS supplement, 0.35 mM L-proline (all from Sigma-Aldrich) and 10 ng/ml TGF-β 3 (Miltenyi Biotech) and cultured for six weeks.…”

“…HSA-HA preparation and cell embedding into hydrogels are described in [34]. Five x 10 5 cells were embedded into 500 μl hydrogel.…”

Quality Control for Mesenchymal Stromal Cells: Chondrogenesis as a Standard Condition?

“…Human serum albumin (HSA) and HSA-HA were produced as described elsewhere [13]. The HSA hydrogel used here was based on a 15 VN primers was used for reverse transcription (RT) according to the manufacturer's recommendations.…”

“…The other situation is the issue of different technical environments for the cells during homing and differentiation. This situation is challenged by comparing radically different concepts: the micromass culture system with cell-cell contact and interactions [4], and hydrogel systems with no cell-cell contact -the gelatin hydrogel with all the bioactive collagen peptides automatically present, and the albumin hydrogel, containing no bioactive signals [13]. In addition, the signaling factor environment is modulated by applying different growth factors, and introducing hyaluronan to provide selective anchorage for CD44 (an MSC-associated cell surface marker) and secondary support such as scavenging oxygenating radicals.…”