Differential Response of Encapsulated Nucleus Pulposus and Bone Marrow Stem Cells in Isolation and Coculture in Alginate and Chitosan Hydrogels

Abstract

Cell-based therapies may hold significant promise for the treatment of early stage degeneration of the intervertebral disc (IVD). Given their propensity to proliferate and ability to form multiple tissue types, mesenchymal stem cells (MSCs) have been proposed as a potential cell source to promote repair of the nucleus pulposus (NP). However, for any successful cell-based therapy a carrier biomaterial may be essential for targeted delivery providing key biophysical and biochemical cues to facilitate differentiation of MSCs. Two widely used biomaterials for NP regeneration are chitosan and alginate. The primary objective of this study was to assess the influence of alginate and chitosan hydrogels on bone marrow (BM) MSCs and NP cells in isolation or in co-culture. A secondary objective of this study was to investigate co-culture seeding density effects of BM and NP cells and simultaneously explore which cell type is responsible for matrix formation in a co-cultured environment. Porcine NP and BM cells were encapsulated in alginate and chitosan hydrogels separately at two seeding densities (4x10 6 and 8x10 6 cells/ml) or in co-culture (1:1, 8 x10 6 cells/ml). Constructs (diameter:5mm, height:3mm) were maintained under IVD-like conditions (low-glucose, low (5%) oxygen) with or without TGF-β3 supplementation for 21 days. Results demonstrated differential viability depending on hydrogel type. NP cells remained viable in both biomaterial types whereas BM viability was diminished in chitosan.Furthermore, hydrogel type was found to regulate sGAG and collagen accumulation.Specifically, alginate better supports sGAG accumulation and collagen type II deposition for both NP and BM cell types compared to chitosan. Having identified that alginate more readily supports cell viability and matrix accumulation we further explored additional effects of seeding density ratios (NP: BM -1:1, 1:2) for co-culture studies. Interestingly, in coculture conditions, the BM cell population declined in number while NP cells increased indicating that MSCs may in fact be signalling NP cells to proliferate rather than contributing to matrix formation. These findings provide exciting new insights on the potential of MSCs for NP tissue regeneration strategies.

Multilineage Constructs for Scaffold‐Based Tissue Engineering: A Review of Tissue‐Specific Challenges

“…An optimization of the cell ratio between chondrocytes and stem cells is however needed for the optimal outcomes of this coculture system. Seeding 2 and 4 times more BMSCs than chondrocytes was more effective than equal quantities in polymer gels . Similarly, a primary human chondrocytes:BMSC ratio of 1:3 leads to the highest chondro‐induction on collagen mesh in vitro .…”

Low‐oxygen conditions promote synergistic increases in chondrogenesis during co‐culture of human osteoarthritic stem cells and chondrocytes

“…There have also been reports of increases in chondrogenic markers through soluble paracrine signalling (Ahmed, Dreier, Göpferich, Grifka, & Grässel, ; Aung, Gupta, Majid, & Varghese, ). One study that investigated the co‐culture of nucleus pulpous (NP) cells and BMSC showed a large increase in the DNA of NP cells while the “supporting” BMSC population decreased in number (Naqvi & Buckley, ). Other studies have observed a similar phenomenon of “chondro‐induction” mediated by two concomitant effects: MSC‐induced chondrocyte proliferation and chondrocyte‐enhanced MSC chondrogenesis (Acharya et al, ).…”

Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments

“…NP cells were harvested from the intervertebral discs (IVDs) of porcine donors ( n = 2, 3–4 months, 20–30 kg) within 3 h of sacrifice as previously described (Naqvi & Buckley, ). NP tissue was isolated and enzymatically digested in 2.5 mg mL −1 pronase solution for 1 h followed by 3 h in 0.5 mg mL −1 collagenase solution at 37 °C.…”