Transforming Growth Factor-Beta 3 Stimulates Cartilage Matrix Elaboration by Human Marrow-Derived Stromal Cells Encapsulated in Photocrosslinked Carboxymethylcellulose Hydrogels: Potential for Nucleus Pulposus Replacement

Abstract

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies using marrow-derived stromal cells (MSCs) have been used to develop cartilaginous tissue constructs, which may serve as viable NP replacements. Supplementation with growth factors, such as transforming growth factor-beta 3 (TGF-β3), has been shown to enhance the differentiation of MSCs and promote functional tissue development of such constructs. A potential candidate material that may be useful as a scaffold for NP tissue engineering is carboxymethylcellulose (CMC), a biocompatible, cost-effective derivative of cellulose. Photocrosslinked CMC hydrogels have been shown to support NP cell viability and promote phenotypic matrix deposition capable of maintaining mechanical properties when cultured in serum-free, chemically defined medium (CDM) supplemented with TGF-β3. However, MSCs have not been characterized using this hydrogel system. In this study, human MSCs (hMSCs) were encapsulated in photocrosslinked CMC hydrogels and cultured in CDM with and without TGF-β3 to determine the effect of the growth factor on the differentiation of hMSCs toward an NP-like phenotype. Constructs were evaluated for matrix elaboration and functional properties consistent with native NP tissue. CDM supplemented with TGF-β3 resulted in significantly higher glycosaminoglycan content (762.69±220.79 ng/mg wet weight) and type II collagen (COL II) content (6.25±1.64 ng/mg wet weight) at day 21 compared with untreated samples. Immunohistochemical analyses revealed uniform, pericellular, and interterritorial staining for chondroitin sulfate proteoglycan and COL II in growth factor-supplemented constructs compared with faint, strictly pericellular staining in untreated constructs at 21 days. Consistent with matrix deposition, mechanical properties of hydrogels treated with TGF-β3 increased over time and exhibited the highest peak stress in stress-relaxation (σ(pk)=1.489±0.389 kPa) at day 21 among all groups. Taken together, these results demonstrate that hMSCs encapsulated in photocrosslinked CMC hydrogels supplemented with TGF-β3 are capable of elaborating functional extracellular matrix consistent with the NP phenotype. Such MSC-laden hydrogels may have application in NP replacement therapies.

In Vitro Characterization of a Stem-Cell-Seeded Triple-Interpenetrating-Network Hydrogel for Functional Regeneration of the Nucleus Pulposus

“…Oxidized dextran and N-carboxyethyl chitosan were synthesized in the laboratory as described previously. 16 Aqueous solutions of 20% teleostean, 3% N-carboxyethyl chitosan, and 7.5% oxidized dextran were mixed at ratio of 1:1:2. The hydrogel was cast between two glass plates and constructs of 4-mm diameter · 2.25-mm thick were produced using a biopsy punch.…”

“…Many promising biomaterials are being investigated, including hyaluronic acid composite hydrogels, [10][11][12][13][14][15] photo-crosslinked carboxymethylcellulose hydrogels, 16,17 and many others. It has been suggested that the ideal hydrogel for NP augmentation and repair would (1) be suitable for minimally invasive (percutaneous) delivery to the NP space, (2) solidify rapidly after implantation to avoid leakage of cells or gel, (3) be capable of restoring disc structure and function, (4) be biologically compatible with the existing NP cell population, and (5) support cell growth and matrix deposition by codelivered cells, including native and stem cells.…”

Mesenchymal Stem Cell in the Intervertebral Disc

“…Three-dimensional (3D) cultures have been used to induce BMSCs to differentiate into chondrocyte-like cells. These include cell pellet, alginate bead, hydrogel, and engineered 3D scaffold [20][21][22][23][24]. Chondrocyte-like phenotype can also be obtained via a single monolayer coculture of BMSCs, either with NPC or annulus fibrosis (AF) cells with cell-cell contact [25,26].…”

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

“…Human MSCs were purified from bone marrow (AllCells, Emeryville, CA, USA) using enriched density centrifugation as previously described (Gupta et al 2011;Chen et al 2013). Briefly, a Human MSC Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada) (Lee et al 2005) was added to the bone marrow and incubated for 20 min to allow for unwanted cells and red blood cells to be crosslinked via tetrameric antibody complexes.…”

“…Carboxymethylcellulose (CMC), a watersoluble polysaccharide derivative of cellulose (Klemm et al 2005), has also been evaluated as an NP tissue scaffold due to its biocompatibility, low-cost, FDAapproval and commercial availability in high purity forms (Ogushi et al 2007). Recently, photocrosslinked CMC hydrogels were shown to support cell viability and the assembly of functional extracellular matrix (ECM) by encapsulated human marrow-derived mesenchymal stromal cells (hMSCs) when cultured in serumfree, chemically defined medium supplemented with TGF-β3 (Gupta et al 2011). However, the mechanical functionality of these constructs did not achieve native levels of the NP (Cloyd et al 2007), providing motivation to refine hydrogel fabrication parameters in order to improve mechanical properties.…”