Proliferation of rat mesenchymal stem cells in collagen sponges reinforced with poly(ethylene terephthalate) fibers by stirring culture method.

The objective of this study is to investigate the effect of medium stirring conditions on the proliferation of rat mesenchymal stem cells (MSC) in collagen sponges reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers. A collagen solution with PET fibers homogeneously dispersed was freeze-dried, followed by dehydrothermal cross-linking to obtain a collagen sponge incorporating PET fibers. MSC were proliferated in the sponge by a stirring culture method. The PET fibers reinforcement significantly suppressed the sponge deformation in culture. The MSC proliferation was enhanced by the stirring culture to a significantly higher extent than that of a static one. Homogeneous distribution of cells proliferated was observed at the stirring rate of 50 rpm and compared with that at lower and higher rates. Combination of the PET fiber-reinforced sponge with the stirring culture method is a promising way to allow cells to homogeneously proliferate in the sponge.

Long-term observation of auto-cell transplantation in non-human primate reveals safety and efficiency of bone marrow stromal cell-derived Schwann cells in peripheral nerve regeneration.

Based on their differentiation ability, bone marrow stromal cells (MSCs) are a good source for cell therapy. Using a cynomolgus monkey peripheral nervous system injury model, we examined the safety and efficacy of Schwann cells induced from MSCs as a source for auto-cell transplantation therapy in nerve injury. Serial treatment of monkey MSCs with reducing agents and cytokines induced their differentiation into cells with Schwann cell properties at a very high ratio. Expression of Schwann cell markers was confirmed by both immunocytochemistry and reverse transcription-polymerase chain reaction. Induced Schwann cells were used for auto-cell transplantation into the median nerve and followed-up for 1year. No abnormalities were observed in general conditions. Ki67-immunostaining revealed no sign of massive proliferation inside the grafted tube. Furthermore, (18)F-fluorodeoxygluocose-positron emission tomography scanning demonstrated no abnormal accumulation of radioactivity except in regions with expected physiologic accumulation. Restoration of the transplanted nerve was corroborated by behavior analysis, electrophysiology and histological evaluation. Our results suggest that auto-cell transplantation therapy using MSC-derived Schwann cells is safe and effective for accelerating the regeneration of transected axons and for functional recovery of injured nerves. The practical advantages of MSCs are expected to make this system applicable for spinal cord injury and other neurotrauma or myelin disorders where the acceleration of regeneration is expected to enhance functional recovery.

Regeneration of anterior cruciate ligament by biodegradable scaffold combined with local controlled release of basic fibroblast growth factor and collagen wrapping.

The objective of this study was to increase the therapeutic efficacy of anterior cruciate ligament (ACL) surgery using an artificial ligament material developed through a combination of tissue engineering technologies. A poly-L-lactic acid (PLLA) scaffold of plain-woven braid was incorporated with a gelatin hydrogel for controlled release of basic fibroblast growth factor (bFGF) and wrapped with a collagen membrane to allow space for ligament regeneration. For the ACL reconstruction surgery, the PLLA braid scaffold combined with the gelatin hydrogel incorporating bFGF and the collagen wrapping was applied to a tunnel prepared in the femur and tibia of rabbits. The hydrogel was placed in the bone, whereas the portion of the braid inside the joint cavity was wrapped with the membrane. As controls, the PLLA scaffold was applied with the hydrogel or the membrane, or without either material. Bone regeneration in the tunnel and ACL tissue regeneration in the joint cavity were histologically evaluated, and the mechanical strength and collagen content of the regenerated ACL were assessed. When the PLLA scaffold was integrated with both the hydrogel and the membrane, bone and ACL tissues were regenerated in the corresponding sites, in marked contrast to the control groups. Combination of bFGF-controlled release resulted in enhanced mechanical strength of the regenerated ACL tissue. In the joint cavity, it is possible that the local bFGF release inside the membrane enhanced the cell migration and collagen production, and that the surrounding PLLA scaffold results in the biological regeneration of ligament-like tissue. Additionally, significant bone regeneration around the scaffold was observed in the bone tunnel. It is therefore possible that the local controlled release of bFGF near the PLLA braid induced both osseointegration and intrascaffold cell migration in the bone tunnel and joint cavity, respectively, resulting in an overall increase in the mechanical strength of the regenerated ACL.

Proliferation, osteogenic differentiation, and distribution of rat bone marrow stromal cells in nonwoven fabrics by different culture methods.

The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpm > the stirred at 10 rpm > the agitated > the static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method.

Cell attachment to PET films coated with a thermo-sensitive block co-polymer with different chemical composition.

This objective of this study is to characterize the surface of poly(ethylene terephthalate) (PET) films coated with the thermo-sensitive di-block co-polymers of 2-ethoxyethyl vinyl ether and 2-phenoxyethyl vinyl ether segments (EOVE-b-PhOVE) with a high polydispersity and evaluate the behavior of cell attachment on them at different temperatures. The EOVE segment possessed a low critical solution temperature at 20 degrees C while the hydrophobic PhOVE segment functioned as the site to allow the co-polymer to adsorb onto the PET films. X-ray photoelectron spectroscopy and contact angle measurements revealed that the PET film was coated with the EOVE-b-PhOVE co-polymers. The density of co-polymers coated increased with the concentration of co-polymers used for coating. Irrespective of the co-polymer type, 3T3L1 cells attached on the surface of coated films at 37 degrees C, while the cells showed a spread shape, which is similar to that of cells attached on the original non-coated film. However, when the temperature decreased from 37 to 4 degrees C, the cell shape changed to be round, in contrast to that of the original PET film. The percent increase of round cells depended on the coating density and the polymerization degree of EOVE segment.

Enhanced proliferation and osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with different poly(ethylene terephthalate) fibers.

The objective of this study was to investigate the feasibility of collagen sponges mechanically reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers in stem cell culture. A collagen solution with homogeneously dispersed PET fibers was freeze-dried, followed by dehydrothermal cross-linking to obtain the collagen sponge incorporating PET fibers. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 200 microm, irrespective of PET fiber incorporation. As expected, PET fibers incorporation significantly enhanced the compression strength of collagen sponge. When used for rat mesenchymal stem cells (MSC), the collagen sponge incorporating PET fibers was superior to the original collagen sponge without PET fibers incorporation in terms of the initial attachment, proliferation and osteogenic differentiation of cells, irrespective of the amount and diameter of fibers incorporated. The shrinkage of sponges during cell culture was significantly suppressed by the fiber incorporation. It is possible that the shrinkage suppression maintains the three-dimensional inner pore structure of collagen sponges without impairing the cell compatibility, resulting in the superior MSC attachment and the subsequent osteogenic differentiation in the sponge incorporating PET fiber.

Preparation of hybrid scaffold from fibrin and biodegradable polymer fiber.

A biodegradable hybrid scaffold was prepared from fibrin and poly(glycolic acid) (PGA) fiber. Mixed fibrinogen and thrombin solution homogeneously dispersed in the presence of various amounts (0, 1.5, 3.0, and 6.0mg) of PGA fiber was freeze-dried to obtain fibrin sponges with or without PGA fiber incorporation. By scanning electron microscopy observation, the fibrin sponges had an interconnected pore structure, irrespective of the amount of PGA fiber incorporated. PGA fiber incorporation enabled the fibrin sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly larger for any fibrin sponge with PGA fiber incorporation than for the fibrin sponge without PGA fiber. The shrinkage of sponges after cell seeding was suppressed by fiber incorporation. It is possible that the shrinkage suppression of sponges maintains their intraspace, resulting in the superior cell attachment of a sponge with PGA fiber incorporation. After subcutaneous implantation into the backs of mice, the residual volume of a fibrin sponge with PGA fiber incorporation was significant compared with that of a fibrin sponge without PGA fiber. Larger number of cells infiltrated deep inside the fibrin sponges with PGA fiber incorporation implanted subcutaneously. It is concluded that the fibrin sponge reinforced by fiber incorporation is a promising three-dimensional scaffold of cells for tissue engineering.

Proliferation and differentiation of rat bone marrow stromal cells on poly(glycolic acid)-collagen sponge.

We studied the effects of dexamethasone (Dex) and basic fibroblast growth factor (bFGF) on proliferation and differentiation of rat bone marrow stromal cells (RBMSCs), using three scaffolds: collagen sponge, poly(glycolic acid) (PGA)-collagen sponge, and PGA-collagen (UV) sponge. RBMSCs were seeded into the sponges, and cultured in primary medium, primary medium with Dex, and primary medium with bFGF and Dex. Three weeks after cultivation, we examined alkaline phosphatase (ALP) activity and cell number in the sponges, and also performed macroscopic, light microscopic, and scanning electron microscopic (SEM) observations. Collagen sponge shrank considerably, but PGA-collagen and PGA-collagen (UV) sponges maintained most of their original shape. PGA-collagen (UV) sponge supplemented with bFGF and Dex together had the highest ALP activity and cell number, followed by PGA-collagen sponge. Although collagen sponge showed cell proliferation only on the surface, the other two sponges showed cell proliferation in the interior. SEM showed the best cell attachment to PGA-collagen (UV) sponge in the presence of bFGF and Dex, followed by PGA-collagen sponge. In conclusion, PGA-collagen (UV) and PGA-collagen sponges proved to be much more useful as scaffolding for bone regeneration when combined with bFGF and Dex.