Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture

Abstract

These findings suggest that while MSCs do generate constructs with substantial cartilaginous properties, further optimization must be done to achieve levels similar to those produced by chondrocytes.

Cartilage Matrix Formation by Bovine Mesenchymal Stem Cells in Three-dimensional Culture Is Age-dependent

“…Likewise, bovine MSCs show a sharp decrease with age in cartilage matrix-forming capacity that is more severe than reported for human MSCs in this same format [5]. Overall, at each age, and under ideal conditions (absence of TGF for chondrocytes, presence of TGF for MSCs), bovine chondrocytes in pellet culture produce more GAG and collagen than MSCs, consistent with our previous findings [17,22,33]. Taken together, when considering an autologous cellbased tissue engineering strategy for cartilage repair, age must be an important consideration.…”

“…Interestingly, fetal and juvenile chondrocyte pellets in chemically defined medium with TGF-b3 accumulated less GAG and collagen (fetal only) than those cultured without TGF-b3, a result that has not been previously reported. In contrast, TGF-b3 improved both GAG content and mechanical properties for immature chondrocytes in the context of three-dimensional agarose hydrogels [33]. This may indicate a microenvironmental influence (such as cell-to-cell contact) in the interpretation of this soluble factor.…”

“…In contrast, we found juvenile bovine MSCs are inferior in terms of functional ECM production to donormatched chondrocytes in various hydrogels [17,23,25,33] but have not considered MSC age in our hyaluronic acid (HA) hydrogel system. Thus, although the literature demonstrates aging affects MSC and chondrocyte function, the relative effects of aging of bovine chondrocytes compared with MSCs is unknown.…”

“…Fetal (second or third trimester; JBS, Souderton, PA), juvenile (3-6 months; Research 87, Boylston, MA), and adult bovine limbs (2-3 years; Animal Technologies, Tyler, TX) were acquired within 24 hours of slaughter. MSCs from three donors of each age were isolated from tibial or femoral bone marrow extractions by plastic adherence [33] and maintained separately in growth medium (DMEM, 10% fetal bovine serum, and 1% penicillinstreptomycin-fungizone; Invitrogen) through Passage 2 or 3. Diced, full-thickness articular cartilage from three donors of each age group was enzymatically digested to release the chondrocytes, which were used without passaging [33].…”

“…Chondrocytes (primary) and bone marrow-derived MSCs (expanded) from fetal, juvenile, and adult bovine donors (three donors per age) were isolated, formed into cell-rich pellets (200,000 per pellet), and cultured for 6 weeks in medium with (CM+) or without (CMÀ) the chondrogenic induction factor transforming growth factorb3 (TGF-b3) [33]. Biochemical assays for DNA, sulfated GAG, and collagen content in each pellet were performed (n = 3 per donor) and proteoglycan and collagen distribution was assessed by histology (Fig.…”