Safety and Efficacy of Kartigen® in Treating Cartilage Defects: A Randomized, Controlled, Phase I Trial.

Here, we aimed to investigate the safety and preliminary efficacy of Kartigen®, a matrix with autologous bone marrow mesenchymal stem cell-derived chondrocyte precursors embedded in atelocollagen. As a surgical graft, Kartigen® was implanted onto the cartilage defects at the weight-bearing site of the medial femoral condyle of the knee. Fifteen patients were enrolled and stratified into two groups, undergoing either Kartigen® implantation (n = 10) or microfracture (control group, n = 5). The primary endpoint was to evaluate the safety of Kartigen® by monitoring the occurrence of adverse events through physician queries, physical examinations, laboratory tests, and radiological analyses for 2 years. There were no infections, inflammations, adhesions, loose body, or tumor formations in the Kartigen®-implanted knees. The preliminary efficacy was assessed using the International Knee Documentation Committee (IKDC) score, visual analog scale, and second-look arthroscopy. The postoperative IKDC scores of the Kartigen® group significantly improved in the 16th week (IKDC = 62.1 ± 12.8, p = 0.025), kept increasing in the first year (IKDC = 78.2 ± 15.4, p < 0.005), and remained satisfactory in the second year (IKDC = 73.6 ± 13.8, p < 0.005), compared to the preoperative condition (IKDC = 47.1 ± 17.0), while the postoperative IKDC scores of the control group also achieved significant improvement in the 28th week (IKDC = 68.5 ± 6.1, p = 0.032) versus preoperative state (IKDC = 54.0 ± 9.1). However, the IKDC scores decreased in the first year (IKDC = 63.5 ± 11.6) as well as in the second year (IKDC = 52.6 ± 16.4). Thirteen patients underwent second-look arthroscopy and biopsy one year after the operation. The Kartigen® group exhibited integration between Kartigen® and host tissue with a smooth appearance at the recipient site, whereas the microfracture group showed fibrillated surfaces. The histological and immunohistochemical analyses of biopsy specimens demonstrated the columnar structure of articular cartilage and existence of collagen type II and glycosaminoglycan mimic hyaline cartilage. This study indicates that Kartigen® is safe and effective in treating cartilage defects.

Partial enzyme digestion facilitates regeneration of crushed nerve in rat.

Peripheral nerve injury is a life-changing disability with significant socioeconomic consequences. In this rat model, we propose that partial enzyme digestion can facilitate the functional recovery of a crushed nerve. The sciatic nerves were harvested and in vitro cultured with the addition of Liberase to determine the appropriate enzyme amount in the hyaluronic acid (HA) membrane. Then, the sciatic nerve of adult male Sprague-Dawley rats was exposed, crushed, and then treated with partial enzyme digestion (either 0.001 or 0.002 unit/mm2 Liberase-HA membrane). The sciatic function index (SFI) for functional recovery of the sciatic nerve was evaluated. After 2 h of in vitro digestion, fascicles and axons were separated from each other, with the cells mobilized. Greater destruction of histology structures occurred in the high enzyme (Liberase-HA membrane at 0.002 unit/mm2) group at 24 h than in the low enzyme (0.001 unit/mm2) group at 48 h. In the SFI evaluation, the improvement in 0.001 unit/mm2 Liberase group was significantly better than control and 0.002 unit/mm2 Liberase group. Our study demonstrated that appropriate enzyme digestion had a significantly faster and earlier recovery.

Newton Output Blocking Force under Low-Voltage Stimulation for Carbon Nanotube-Electroactive Polymer Composite Artificial Muscles.

This is a study on the development of carbon nanotube-based composite actuators using a new ionic liquid-doped electroactive ionic polymer. For scalable production purposes, a simple hot-pressing method was used. Carbon nanotube/ionic liquid-Nafion/carbon nanotube composite films were fabricated that exhibited a large output blocking force and a stable cycling life with low alternating voltage stimuli in air. Of particular interest and importance, a blocking force of 1.5 N was achieved at an applied voltage of 6 V. Operational durability was confirmed by testing in air for over 30 000 cycles (or 43 h). The superior actuation performance of the carbon nanotube/ionic liquid-Nafion/carbon nanotube composite, coupled with easy manufacturability, low driving voltage, and reliable operation, promises great potential for artificial muscle and biomimetic applications.

Cardiac repair using chitosan-hyaluronan/silk fibroin patches in a rat heart model with myocardial infarction.

The cardiac repair of myocardial infarction (MI) hearts of rats using chitosan-hyaluronan/silk fibroin (chitosan-HYA/SF) cardiac patches was examined after eight weeks of implantation. Rats with implantations of chitosan-HYA/SF patches (CHS group) significantly (P<0.05) reduced the dilation of the inner diameter of left ventricle (LV) (4.27 ± 0.29 mm), increased wall thickness of LV (1.5 ± 0.13 mm) and improved the fractional shortening of LV of hearts (LVFS) (42.8 ± 2.4%) compared with those values of LVs of rats without implants (MI group) (e.g., 5.92 ± 0.39 mm, 1.2 ± 0.06 mm and 31.5±1.4%, respectively). Moreover, blood vessel-like structures in MI regions of LVs in the CHS group were widely distributed while none was found in the MI group. The CHS group significantly improved the secretion of paracrine factors, such as VEGF in the MI regions of LVs (P<0.05, n=4), relative to that in the MI group. In conclusion, chitosan-HYA/SF cardiac patches are promising biomaterials for the cardiac repair of MI rat hearts.

Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model.

Bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid (BMSC/SH) patches were implanted into myocardial infarction (MI) rat hearts to investigate the efficacies of them on enhancing left ventricular (LV) remodeling and cardiac repair. 45 rats were divided into four groups: Sham, MI (MI hearts, induced by a cryo-injury technique), SH and BMSC/SH (MI hearts with implantations of SH and BMSC/SH patches, respectively). After eight weeks of post-implantation, the patches for the SH and BMSC/SH groups were intact and well adhered on the MI zones with no and minor immunological responses, respectively, examined by a CD68 marker, while severe inflammation on the zones was observed for the MI group. The SH group showed the efficacy of cardiac repair on MI zones. Moreover, BMSC/SH group significantly improved the wall thickness of LV, assessed by echocardiography, and had high viability of delivery BMSC, largely reduced apoptosis, significantly promoted neo-vascularization and stimulated the secretions of various paracrine factors such as VEGF, examined by real-time PCR, in MI zones compared with those of the SH and MI groups. In conclusion, the therapeutic efficacies of using BMSC/SH patches for repairing MI hearts were demonstrated by showing the advantages of both bioactive SH patches and BMSC-based therapy.

Promoting regeneration of peripheral nerves in-vivo using new PCL-NGF/Tirofiban nerve conduits.

Poly(ε-caprolactone) (PCL) scaffolds were modified by grafting nerve growth factor (NGF) and Tirofiban (TF), a clinical anti-thrombosis drug, as a new biomaterial for producing nerve conduits to promote the regeneration of sciatic nerves. The successful grafting of NGF and TF onto PCL scaffolds was confirmed by FTIR and ESCA spectra. In-vitro growths of the PC12 cells in PCL-NGF and PCL-NGF/TF scaffolds, determined by MTS, were significantly higher (P < 0.05, n = 4) than those in the PCL scaffolds following three days of cultivation. Interestingly, this study evaluation of the PCL, PCL-NGF, and PCL-NGF/TF nerve conduits in a 12 mm long gap of the rat sciatic nerve defect model that the gastrocnemius muscle mass of the tested rats in the PCL-NGF/TF groups significantly exceeded those in the PCL-NGF and PCL group. In the rats that had been implanted with PCL-NGF/TF conduits, the generated nerves passed through those conduits, expressing beta-III tubulin (TB), growth association protein-43 (GAP-43) and myelin basic protein (MBP) along their longitudinal axis, and the proximal and distal nerve ends of the rats were successfully connected. Those that had been implanted with PCL and PCL-NGF conduits did not exhibit these effects, as revealed by an immunochemical study of the expressions of the proteins in the conduits. Moreover, counting within the dorsal horn of the spinal cord (C(5)) demonstrated that the numbers of CTB-HRP-labeled neurons in the rats that had been implanted with PCL-NGF/TF conduits were significantly higher than those in the other groups. In this study, in-vivo examinations of the use of newly designed PCL-NGF/TF conduits to promote the generation of nerves in a defective rat model significantly increased the gastrocnemius muscle mass, and led to the successful regeneration of nerves that bridged a 12 mm long defected gap of nerves in rats. However, more rats must be tested to confirm the efficacy the newly designed nerve conduits.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin - Hyaluronic acid cardiac patches.

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin-polysaccharide cardiac patches in vitro.

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS-hyaluronic acid (SF/CS-HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR-FTIR spectra confirm the co-existence of CS or CS-HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS-HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded (P<0.001) that on culture wells after seven days of cultivation. Additionally, the relative growth rates of rMSCs on SF/CS and SF/CS-HA hybrid patches were significantly better (P<0.01) than that on SF patches that were also observed by using vimentin stain to the cells. For instance, the relative cell growth rates (%) in cell culture wells, SF, SF/CS and SF/CS-HA patches were 100%, 282.9+/-6.5%, 337.0+/-8.0% and 332.6+/-6.6% (n=6, for all), respectively. For investigating the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real-time quantitative polymerase chain reaction (real-time PCR) analysis. The results of cardiomyogenic differentiation of induced rMSCs on SF/CS and SF/CS-HA hybrid patches significantly improved the expressions of cardiac genes of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) than those on SF patches and culture wells. Interestingly, the results of cardiac gene expressions of the cells on the SF/CS-HA hybrid patches were the most pronounced in promoting cardiomyogenic differentiations in this investigation. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS and SF/CS-HA hybrid patches were much pronounced compared with SF patches, indicating the improvements of cardiomyogenic differentiation on the hybrid patches. The results of this study demonstrate that the SF/CS and SF/CS-HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues.