A Comparison of Three-Dimensional Culture Systems to EvaluateIn VitroChondrogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

Objective: To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5 · 10 5 cell pellets, 5 · 10 5 cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-b1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X). Results: alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5 · 10 5 cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels. Conclusion: equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems.

RGD‐functionalized polyethylene glycol hydrogels support proliferation and in vitro chondrogenesis of human periosteum‐derived cells

“…A 3D environment, via aggregation or pellet structures, has been proven to be crucial for the differentiation of MSCs down the chondrogenic lineage . However, pellet structures tend to guide the MSC population toward hypertrophy, an undesired outcome that has been shown to be circumvented through the use of a 3D hydrogel system . In addition to providing a direct means of creating a sufficiently sized repair construct, hydrogels mimic the native environment of the cells and can be modified to the cells' specific needs, reducing the divide between 2D cell culture and in vivo conditions .…”

Chitosan‐agarose scaffolds supports chondrogenesis of Human Wharton's Jelly mesenchymal stem cells

“…The gene expression of the chondrogenic markers, COL2A1, ACAN, SOX6, and SOX9 indicate that the CHAG scaffolds supported chondrogenic differentiation of HWJ‐MSCs . The reduced gene expression of COL10A1 suggests that HWJ‐MSCs are remaining in a more hyaline‐like differentiated state, while limiting the terminal differentiation toward hypertrophy . Hypertrophic chondrocytes induces mineralization and in turn leads to inferior mechanical properties and function of the cartilage .…”

“…27,28 The reduced gene expression of COL10A1 suggests that HWJ-MSCs are remaining in a more hyaline-like differentiated state, while limiting the terminal differentiation toward hypertrophy. 43 Hypertrophic chondrocytes induces mineralization and in turn leads to inferior mechanical properties and function of the cartilage. 44 The moderate staining of safranin-O indicates the complete attachment of the cells and the presence of proteoglycans synthesized by the cells.…”

State of the art: Stem cells in equine regenerative medicine

“…Hence totipotent cells occur only in early stages of embryonic development, pluripotent in later embryonic and early fetal stages, multipotent in late fetal and fully developed (adult) tissue stages, and unipotent, sometimes called progenitor, cells only in adult stages. Tissue harvest sites for undifferentiated equine cells from fully developed tissue include adipose tissue [28,[42][43][44][45][46][47][48], bone marrow [1][2][3][4][5]7,11,22,34,[49][50][51][52][53][54][55][56], peripheral blood [14,35,57], amnion [13,53], amniotic fluid [58], umbilical cord tissue [40,59], umbilical cord blood [29,48,[60][61][62], tendon [63], muscle [28,63], periosteum [28] and cornea [36] (Fig 1, Table S1). Cells have also been isolated from equine embryonic [64]…”

“…Loss of normal musculoskeletal tissue function can be career-and, potentially, life-ending in horses. It is no surprise that tissue targets of equine regenerative medicine include cartilage [1][2][3][4][5][6][7], tendon/ligament [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24], bone [25][26][27][28][29][30][31][32] hoof lamina [33] and meniscus [34]. Equine skin [35] and cornea [36], among others, have also been considered for regenerative therapy.…”