Development of functional reconstructive surgery in cranio-maxillo-facial area using tissue engineering

我们证明了聚合物支架中再生软骨的中空结构，并推测不成熟的结构与通过聚合物结构和硬的新软骨的营养流入的阻塞有关。换句话说，氧气和营养物质对于在聚合物支架的深层内存活和维持接种细胞至关重要。为了解决这个问题，我们研究了一种可生物降解的天然聚合物相比，传统的合成生物降解聚合物。天然聚合物的片材显示出比常规聚合物片材显著更好的氧气和营养物渗透性。为了产生三维形状，使用有机溶剂浇铸特定形状的蔗糖颗粒。然而，由于有机溶剂即使在完全蒸发后也不够安全用于临床应用，我们开发了一种使用盐和水的新方法。支架表面及中心均保持海绵状结构，软骨基质再生。然而，体外软骨再生需要长时间培养，至少需要几个月，骨髓基质细胞（BMSC）的增殖和分化面临许多困难。BMSC需要维持高细胞密度，即使在原代培养增殖。除非保持高的细胞密度，否则细胞形态变得平坦和宽。这意味着BMSC分化并停止增殖。其他研究表明，软骨再生所需的时间和生长因子依次为BMSC、软骨膜内的祖细胞和软骨内的软骨细胞。因此，应用体外再生技术进行骨髓间充质干细胞的临床应用应考虑经济条件。

We demonstrated the hollow structure of regenerated cartilage in polymer scaffolds and speculated that immature structure was related to blockage of inflow of nutrients through polymer structure and hard neo-cartilage. In other words, oxygen and nutrients are critical for surviving and maintaining of seeded cells inside the deep layer of the polymer scaffold. To solve this problem, we investigated a biodegradable natural polymer compared to conventional synthetic biodegradable polymers. A sheet of the natural polymer showed significantly better permeability of oxygen and nutrients than a sheet of conventional polymer. To create three-dimensional shapes, organic solvent was used to cast specific shaped sucrose particles. However, since organic solvent was not safe enough even after complete evaporation for clinical applications, we have developed a new procedure using salt and water. Sponge structure was maintained and cartilaginous matrix was regenerated not only near the surface area, but also in the deep center of scaffolds. However, in vitro cartilage regeneration required long term culture at least few months.We faced many difficulties on proliferation and differentiation of bone marrow derived stromal cells (BMSC). BMSC required maintaining high cell density even after primary culture for proliferation. Unless high cell density was kept, cellular morphology became flat and wide. That means BMSC were differentiated and stopped proliferating. Other studies showed that more and more time and growth factors were required for cartilaginous regeneration in order of BMSC, progenitor cells in cartilage membrane and chondrocytes inside cartilage. Therefore we should consider economical condition for clinical application of BMSC using in vitro regeneration procedures.