Biochemical and Structural Characterization of Neocartilage Formed by Mesenchymal Stem Cells in Alginate Hydrogels

Abstract

A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies.

Second harmonic generation imaging in tissue engineering and cartilage pathologies

“…The cells were cultured at appropriate conditions for differentiation down the chondrocyte lineage, see Olderøy et al for details. 4 Standard paraffin sections were prepared. Some sections were stained with antibodies against collagen II, while adjacent sections were only stained with DAPI for SHG imaging.…”

“…3 One strategy is to grow mesenchymal stem cells (MCS) in an alginate scaffold and make the cells differentiate into chondrocytes by appropriate growth factors and metabolic cues. 4 The differentiated MSCs start to produce extracellular matrix components (collagen II and proteoglycans) with the goal of creating a tissue implant with properties resembling hyaline cartilage. The implant can then be shaped according to the lesion and implanted into the knee.…”

Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation

“…Some more recent strategies to partially circumvent these limitations include the use of freeze-dried, CI saturated hydrogels, which when hydrated and mixed with polymer solution will slowly rehydrate and release gelling ions. 9,10 Bulk gels can be formed with limited control over gelling kinetics, however this method still relies on the use of a solid component. CI may also be chelated by a photo-labile protecting group, therefore allowing gelation upon exposure to light (typically near UV) enabling rapid, triggerable gelation kinetics, however release is dependent on the penetration of light making injection into deep or inaccessible areas not possible.…”

Application of Sodium Alginate Hydrogel

“…The existence of RGD peptides in the gels of alginate prohibits a regular phenotype of interacting myoblasts over going interactions [58],osteoblasts, chondrocytes [59,60], [61], follicle of the ovary [62], in addition to the stromal cells of the bone marrow [63][64].such as , the proliferation as well as adhesion of the cultured myoblasts on the gels of alginate were highly improved within conjugation of RGD peptides chemical products to the backbone profile of the alginate, in correlation to the gels of alginate that did not undergo modification (Fig. 13) [64].…”