Bornes Troy D, Jomha Nadr M, Mulet-Sierra Aillette, Adesida Adetola B
Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Li Ka Shing Centre for Health Research Innovation, Divisions of Orthopaedic Surgery and Surgical Research, Department of Surgery, University of Alberta, Edmonton, AB, T6G 2E1, Canada.
Stem Cell Res Ther. 2015 Apr 23;6(1):84. doi: 10.1186/s13287-015-0075-4.
The quality of cartilaginous tissue derived from bone marrow mesenchymal stromal stem cell (BMSC) transplantation has been correlated with clinical outcome. Therefore, culture conditions capable of modulating tissue phenotype, such as oxygen tension and scaffold composition, are under investigation. The objective of this study was to assess the effect of hypoxia on in vitro BMSC chondrogenesis within clinically approved porous scaffolds composed of collagen and hyaluronic acid (HA). It was hypothesized that hypoxic isolation/expansion and differentiation would improve BMSC chondrogenesis in each construct.
Ovine BMSCs were isolated and expanded to passage 2 under hypoxia (3% oxygen) or normoxia (21% oxygen). Cell proliferation and colony-forming characteristics were assessed. BMSCs were seeded at 10 million cells per cubic centimeter on cylindrical scaffolds composed of either collagen I sponge or esterified HA non-woven mesh. Chondrogenic differentiation was performed in a defined medium under hypoxia or normoxia for 14 days. Cultured constructs were assessed for gene expression, proteoglycan staining, glycosaminoglycan (GAG) quantity, and diameter change.
Isolation/expansion under hypoxia resulted in faster BMSC population doublings per day (P <0.05), whereas cell and colony counts were not significantly different (P = 0.60 and 0.30, respectively). Collagen and HA scaffolds seeded with BMSCs that were isolated, expanded, and differentiated under hypoxia exhibited superior aggrecan and collagen II mRNA expressions (P <0.05), GAG quantity (P <0.05), and proteoglycan staining in comparison with normoxia. GAG/DNA was augmented with hypoxic isolation/expansion in all constructs (P <0.01). Comparison by scaffold composition indicated increased mRNA expressions of hyaline cartilage-associated collagen II, aggrecan, and SOX9 in collagen scaffolds, although expression of collagen X, which is related to hypertrophic cartilage, was also elevated (P <0.05). Proteoglycan deposition was not significantly improved in collagen scaffolds unless culture involved normoxic isolation/expansion followed by hypoxic differentiation. During chondrogenesis, collagen-based constructs progressively contracted to 60.1% ± 8.9% of the initial diameter after 14 days, whereas HA-based construct size was maintained (109.7% ± 4.2%).
Hypoxic isolation/expansion and differentiation enhance in vitro BMSC chondrogenesis within porous scaffolds. Although both collagen I and HA scaffolds support the creation of hyaline-like cartilaginous tissue, variations in gene expression, extracellular matrix formation, and construct size occur during chondrogenesis.
源自骨髓间充质基质干细胞（BMSC）移植的软骨组织质量与临床结果相关。因此，能够调节组织表型的培养条件，如氧张力和支架组成，正在研究中。本研究的目的是评估缺氧对在临床批准的由胶原蛋白和透明质酸（HA）组成的多孔支架内体外BMSC软骨生成的影响。假设缺氧分离/扩增和分化将改善每种构建体中的BMSC软骨生成。
绵羊BMSC在缺氧（3%氧气）或常氧（21%氧气）条件下分离并扩增至第2代。评估细胞增殖和集落形成特征。将BMSC以每立方厘米1000万个细胞的密度接种在由I型胶原海绵或酯化HA无纺布组成的圆柱形支架上。在缺氧或常氧条件下，在特定培养基中进行软骨分化14天。对培养的构建体进行基因表达、蛋白聚糖染色、糖胺聚糖（GAG）含量和直径变化评估。
缺氧条件下的分离/扩增导致BMSC每天的群体倍增更快（P<0.05），而细胞和集落计数无显著差异（分别为P = 0.60和0.30）。与常氧相比，接种了在缺氧条件下分离、扩增和分化的BMSC的胶原蛋白和HA支架表现出更高的聚集蛋白聚糖和胶原蛋白II mRNA表达（P<0.05）、GAG含量（P<0.05）和蛋白聚糖染色。在所有构建体中，缺氧分离/扩增使GAG/DNA增加（P<0.01）。按支架组成比较表明，胶原蛋白支架中透明软骨相关的胶原蛋白II、聚集蛋白聚糖和SOX9的mRNA表达增加，尽管与肥大软骨相关的胶原蛋白X的表达也升高（P<0.05）。除非培养包括常氧分离/扩增后再进行缺氧分化，否则胶原蛋白支架中的蛋白聚糖沉积没有显著改善。在软骨生成过程中，基于胶原蛋白的构建体在14天后逐渐收缩至初始直径的60.1%±8.9%，而基于HA的构建体大小保持不变（109.7%±4.2%）。
缺氧分离/扩增和分化增强了多孔支架内体外BMSC软骨生成。虽然I型胶原和HA支架都支持形成透明样软骨组织，但在软骨生成过程中基因表达、细胞外基质形成和构建体大小会发生变化。
