Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair

Abstract

BackgroundCell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.MethodsA hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.ResultsThe reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization.ConclusionsThe data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

Physioxia Expanded Bone Marrow Derived Mesenchymal Stem Cells Have Improved Cartilage Repair in an Early Osteoarthritic Focal Defect Model

“…For the post-trauma model ( Figure 2 a), defects were created on both medial condyles of right and left knee of each rabbit and then either left empty (group 1; n = 6 rabbits) or injected with a commercially available hyaluronic acid hydrogel (TETEC, Tübingen, Germany) [ 32 ] on the contralateral medial femoral condyle without cells (group 2; n = 6). In the case of the focal early OA model ( Figure 2 b), two surgeries were performed, the first involved defect creation as described and then following development of focal early OA, a second surgery was undertaken to clean the defect using a 22G needle to remove repair tissue.…”

“…In the case of MSC treated defects for both models, autologous rabbit MSCs (passage 2; n = 26, including donors from the in vitro study (Section 2.2)) isolated from bone marrow were pre-expanded in parallel under either hyperoxia or physioxia (see Section 2.2). MSCs were counted using a haemacytometer and seeded into the hydrogel at a density of 50 × 10 6 cells/mL according to a previously described protocol [32]. The cell seeding density was based on previous in vivo experiments using MSCs for cartilage repair in animal models [33,34].…”

Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside

“…At T = 0 months, MRI analysis was performed to determine the degree of IVD degeneration. Thereafter, the IVDs in which no NX was performed (noNX-IVDs; five per dog) and the IVDs in which NX was performed (NX-IVDs; five per dog) were either not injected (control) or injected on the right side of the spine with 50 µL of (a) 1 × 10 6 MSCs in a canine albumin-hyaluronan hydrogel [ 48 ] (b) 10 mg/mL NCM, or (c) 1 × 10 6 MSCs suspended in 10 mg/mL NCM. At T = 3 months, MRI analysis was performed and two IVDs per dog (L6-L7 (noNX-IVD) and L7-S1 (NX-IVD), which previously received 50 µL NCM) were reinjected with 50 µL of 10 mg/mL NCM (2x NCM), to test whether a better effect could be achieved with multiple injections.…”

Tissue Engineering Strategies for Intervertebral Disc Treatment Using Functional Polymers

“…Hydrogel made of albumin crosslinked PEG including human IVD cells were experimented in in vitro and in vivo [164]. PEG hydrogel containing IVD cells showed cartilage and IVD specific mRNA, and cell morphology was more stable than monolayer cells.…”