Autologous Synovial Mesenchymal Stem Cell Transplantation Suppresses Inflammation Caused by Synovial Harvesting and Promotes Healing in a Micro Minipig Repaired Meniscus Model.

PURPOSE: Allogeneic synovial mesenchymal stem cells (MSCs) effectively promote meniscus healing in micro minipigs. We investigated the effect of autologous synovial MSC transplantation on meniscus healing in a micro minipig model of meniscus repair showing synovitis after synovial harvesting. MATERIALS AND METHODS: Synovium was harvested from the left knee of the micro minipigs after arthrotomy and used to prepare synovial MSCs. The left medial meniscus in the avascular region was injured, repaired, and transplanted with synovial MSCs. First, synovitis was compared after 6 weeks in knees with and without synovial harvesting. Second, the repaired meniscus was compared for the autologous MSC group and the control group (in which synovium was harvested but MSCs were not transplanted) 4 weeks after transplantation. RESULTS: Synovitis was more severe in knees subjected to synovium harvesting than in knees not subjected to harvesting. Menisci treated with autologous MSCs showed no red granulation at the tear of the meniscus, but menisci not treated with MSCS showed red granulation. Macroscopic scores, inflammatory cell infiltration scores, and matrix scores assessed by toluidine blue staining were all significantly better in the autologous MSC group than in the control group without MSCs (n = 6). CONCLUSION: Autologous synovial MSC transplantation suppressed the inflammation caused by synovial harvesting in micro minipigs and promoted healing of the repaired meniscus.

Photoiniferter-based thermoresponsive graft architecture with albumin covalently fixed at growing graft chain end.

The aim of this study was to develop a novel surface graft architecture in which albumin is covalently fixed at the growing chain end of the hydrophilic polymers: poly(N, N-dimethylacylamide), PDMAM, and poly(N-isopropylacrylamide), PNIPAM. Photoiniferter-based surface-grafted polymers were prepared using either an albuminated iniferter or a nonalbuminated iniferter, both of which were derivatized on glass surfaces, and ultraviolet (UV)-light-irradiated in the presence of a DMAM or NIPAM monomer. Surface chemical composition analysis by X-ray photoelectron spectroscopy, contact angle measurement, immunostaining using fluorescence labeled antibody and the measurement of graft thickness, as determined from force-distance curves obtained in water at 25 degrees C and 37 degrees C by atomic force microscopy, evidenced that the thickness of graft layer increased with photoirradiation time and albumin molecules exist at growing chain ends. For PNIPAM-grafted surfaces, the interconversion between swollen and collapsed graft chains was observed below and above the lower critical solution temperature of PNIPAM. The potential application of a thermoresponsive graft with albumin covalently fixed at its growing chain end was discussed in terms of "active" nonfouling surface design based on the temperature-dependent switching of phase transition.

Poly(N-isopropylacrylamide) (PNIPAM)-grafted gelatin as thermoresponsive three-dimensional artificial extracellular matrix: molecular and formulation parameters vs. cell proliferation potential.

A series of poly(N-isopropylacrylamide)-grafted gelatins (PNIPAM gelatins) of three different graft densities (approx. 11, 22 and 34 graft chains per gelatin molecule) and three different molecular weights of their graft chains (molecular weight approximately 1.2 x 10(4), 5.0 x 10(4) and 1.3 x 10(5) g/mol) were prepared by multiple derivatization of dithiocarbamyl (DC) group in a gelatin molecule and subsequent iniferter (acts as an initiator, transfer-agent and terminator)-based photopolymerization of NIPAM. The weight ratio of PNIPAM graft chains to gelatin (P/G) varied from 1.4 to 49. Aqueous solutions of PNIPAM-gelatins showed thermo-responsiveness, depended on the graft density and the molecular weight of PNIPAM graft chain or P/G. Aqueous solutions (10 or 20%, w/v) of PNIPAM-gelatins with P/G of more than 5.8 were converted to gels at 37 degrees C. Focal plane images of PNIPAM-gelatin gels by confocal laser scanning microscopy revealed that the size of hydrophobically clustered aggregates increased with P/G, whereas the space of microvoids decreased with concentration. Compressive strain-stress measurements revealed that compressive strength of PNIPAM-gelatin increased with P/G. Bovine smooth muscle cells (SMCs)-entrapped gels were produced from PNIPAM-gelatin-containing cell-suspended medium solutions at 37 degrees C. The entrapped cells proliferated in the gel with P/G of more than 12. A higher cell proliferativity was obtained at low concentration (5%, w/v) and higher P/G (>18). Tissue formation composed of proliferative SMCs and cell-secreted extracellular matrices (collagen) was obtained at 14 days incubation. The inter-relationship between the molecular parameters of PNIPAM-gelatin, internal structural features and cell proliferation potential was discussed.

The potential of poly(N-isopropylacrylamide) (PNIPAM)-grafted hyaluronan and PNIPAM-grafted gelatin in the control of post-surgical tissue adhesions.

Poly(N-isopropylacrylamide)-grafted hyaluronan (PNIPAM-HA) and PNIPAM-grafted gelatin (PNIPAM-gelatin), which exhibit sol-to-gel transformation at physiological temperature, were applied as control of tissue adhesions: tissue adhesion prevention material and hemostatic aid, respectively. The rat cecum, which was abraded using surgical gauze, was coated with PNIPAM-HA-containing PBS (concentration: 0.5 w/v%). The coated solution was immediately converted to an opaque precipitate at body temperature, which weakly adhered to and covered the injured rat cecum. One week after coating, tissue adhesion between the PNIPAM-HA-treated cecum and adjacent tissues was significantly reduced as compared with that between non-treated tissue and adjacent tissues. On the other hand, the coating of bleeding spots of a canine liver with PNIPAM-gelatin-containing PBS (concentration: 20 w/v%) resulted in spontaneous gel formation on the tissues and subsequently suppressed bleeding. Although these thermoresponsive tissue adhesion prevention and hemostatic materials are still prototypes at this time, both thermoresponsive biomacromolecules bioconjugated with PNIPAM, PNIPAM-HA and PNIPAM-gelatin, may serve as a tissue adhesion prevention material and hemostatic aid, respectively.

Poly(N-isopropylacrylamide) (PNIPAM)-grafted gelatin hydrogel surfaces: interrelationship between microscopic structure and mechanical property of surface regions and cell adhesiveness.

Poly(N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin) serves as a temperature-induced scaffold at physiological temperature. This study was aimed at determining the effect of the graft architecture of thermoresponsive PNIPAM-gelatin on the surface topography and elastic modulus of the hydrogels prepared with different architectured PNIPAM-gelatins: the surface topography and elastic modulus were determined by atomic force microscopy (AFM). PNIPAM-gelatin surfaces showed an irregularly concavo-convex structure with a vertical interval of approximately 1 microm regardless of the weight ratio of PNIPAM to gelatin (P/G: 5.8, 12, and 18). The elastic moduli of hydrogels varied at measured sites. The mean elastic moduli of PNIPAM-gelatin with the lowest P/G were low, but increased with increasing P/G. Human umbilical vein endothelial cells adhered and spread on PNIPAM-gelatin hydrogels with the highest P/G, whereas reduced adhesion and nonspreading, round-shaped cells resided on the hydrogels with lower P/Gs. Interrelationship between elastic modulus and cell adhesion and spreading potentials were discussed from physicochemical and cellular biomechanical viewpoints.

Three-dimensional cardiac tissue engineering using a thermoresponsive artificial extracellular matrix.

The purpose of this study was to try to reconstitute three-dimensional cardiac tissue using a thermoresponsive artificial extracellular matrix, poly (N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin), as the scaffold. PNIPAM-gelatin solution gels almost immediately when heated above 34 degrees C. We thought this property could become advantageous as scaffolding for reconstituting three-dimensional tissue. Because PNIPAM-gelatin solution gels so quickly, all seeded cells in PNIPAM-gelatin solution would become entrapped and uniformly distributed toward three dimensions. Thus it would be possible to reconstitute three-dimensional tissue by a very simple method of mixing cells and PNIPAM-gelatin solution. Fetal rat cardiac cells were mixed with PNIPAM-gelatin solution, incubated at 37 degrees C to allow the mixture to gel, and cultured in vitro. To define suitable culture conditions the following parameters were tested: (1) PNIPAM-gelatin concentration, 0.04 approximately 0.125 mg/ml; (2) cell seeding density, 1 approximately 50 x 10(6) cells/ml; and (3) addition or not of hyaluronic acid. With a PNIPAM-gelatin concentration of 0.05 mg/ml, a cell seeding density of 50 x 10(6) cells/ml, and the addition of hyaluronic acid, tissue was reconstituted and it contracted synchronously. After hematoxylin and eosin staining, the cells reconstituted three-dimensional tissue, and the tissue cross-section was approximately 60 microm thick.

In vivo evaluation of poly(N-isopropylacrylamide) (PNIPAM)-grafted gelatin as an in situ-formable scaffold.

We examined whether poly(N-isopropylacrylamide)-grafted gelatin (PNIPAM-gelatin) with a lower critical solution temperature of approximately 34 degrees C, which was prepared by quasi-living radical graft polymerization, can serve as an in situ-formable three-dimensional extracellular matrix or cell scaffold. A mixture of fibroblasts stained with fluorescent dye and PNIPAM-gelatin in Dulbecco's modified Eagle's medium solution was injected into the subcutaneous tissue of Wistar rats, and immediately formed a white, opaque cell-incorporated gel. Fibroblasts immediately after injection were spherical in shape and were homogeneously distributed in the gel. Fibroblasts in the gel 2 weeks after injection had spread and proliferated. One day after injection, many macrophages and neutrophiles were observed around the gel. As the implantation period proceeded, the inflammation reaction subsided. One week after injection, fibroblasts in the native tissue and macrophages migrated into the gel. From 6 to 12 weeks after injection, some degree of calcification in the solid tissue was intermittently observed. The weight of the gel 6 weeks after implantation was reduced to almost one-half of the weight of the originally injected sample. The potential usefulness of PNIPAM-gelatin as an injectable scaffold is discussed.

A novel photocurable insulator material for autonomic nerve activity recording.

The two-component, addition-curing silicone glue is widely used as an insulator for autonomic nerve activity recording. Due to its high fluidity before curing, a sizable mass of the glue is needed to completely cover the electrode tips, which may cause mechanical stress on the nerve. To overcome this problem, we designed a novel photocurable insulator material composed of Vaseline and 1,12-dodecanediol diacrylate (50:50wt%) together with a photoinitiator, camphorquinone, at 0.25wt%. This material had an appropriate viscosity of 0.18 Pa s at 25 degrees C and was converted to a soft solid upon an arbitrary timing of photoirradiation. The compressive force per mm deformation of the resulting solid was 155.5 kPa at 1 min of photoirradiation. The impedance of the solid for 1 mm length and 10 mm2 cross-sectional area was above 1 Mohm. In anesthetized rabbits, a very small mass of the photocurable material was able to cover the electrode tips and the nerve in situ. Changes in both the aortic depressor nerve activity and renal sympathetic nerve activity were stably recorded. These results indicate that the photocurable material developed is useful as an in vivo insulator material for autonomic nerve activity recording.