In vitro enhancement and functional characterization of neurite outgrowth by undifferentiated adipose-derived stem cells.

Adipose­derived stem cells (ASCs) can easily be obtained and expanded in vitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted ASCs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated ASCs on nerve regeneration is currently only partially understood. In the present study, undifferentiated ASCs and cutaneous primary afferent dorsal root ganglion (DRG) neurons were co­cultured in order to investigate their interaction. ASCs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co­culture system with DRG cells was established to determine the effect of undifferentiated pluripotent ASCs on neurite elongation. Neurite outgrowth, length and number was examined in the co­culture and compared with single­culture cells and cells stimulated with nerve growth factor (NGF). In ASC cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers CD44, CD73 and CD90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASC differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of DRG neurons was enhanced following co­culture with ASCs, resulting in increased neurite length after 24 h of cultivation. Furthermore, neurite outgrowth of DRG neurons was directed towards the undifferentiated ASC and direct cell­to­cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASC optimizing tissue­engineered constructs may be promising for peripheral nerve repair.

Influence of MMP inhibitor GM6001 loading of fibre coated polypropylene meshes on wound healing: Implications for hernia repair.

Polypropylene meshes are standard for hernia repair. Matrix metalloproteinases play a central role in inflammation. To reduce the inflammatory response and improve remodelling with an associated reduction of hernia recurrence, we modified polypropylene meshes by nanofibre coating and saturation with the broad-spectrum matrix metalloproteinase inhibitor GM6001. The aim was to modulate the inflammatory reaction, increase collagen deposition and improve mesh biointegration. Polypropylene meshes were surface-modified with star-configured NCO-sP(EO -stat-PO) and covered with electrospun nanofibres (polypropylene-nano) and GM6001 (polypropylene-nano-GM). In a hernia model, defects were reconstructed with one of the meshes. Inflammation, neovascularization, bio-integration, proliferation and apoptosis were assessed histologically, collagen content and gelatinases biochemically. Mesh surface modification resulted in higher inflammatory response compared to polypropylene. Pro-inflammatory matrix metalloproteinase-9 paralleled findings while GM6001 reduced matrix metalloproteinase-9 significantly. Significantly increased matrix metalloproteinase-2 beneficial for remodelling was noted with polypropylene-nano-meshes. Increased vascular endothelial growth factor, neo-vascularization and collagen content were measured in polypropylene-nano-meshes compared to polypropylene. GM6001 significantly reduced myofibroblasts. This effect ended after d14 due to engineering limitations with release of maximal GM6001 loading. Nanofibre-coating of polypropylene-meshes confers better tissue vascularization to the cost of increased inflammation. This phenomenon can be only partially compensated by GM6001. Future research will enable higher GM6001 uptake in nano-coated meshes and may alter mesh biointegration in a more pronounced way.

Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo.

Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng/ml) in the absence or presence of the nonselective MMP inhibitor GM6001 for 8 days. The basal culture conditions promoted type I collagen catabolism that was accelerated by TNF-α (p<0.005) and accomplished by MMPs (p<0.005). Levels of the collagenases MMP-8 and MMP-13 were insignificant and neither MMP-2 nor MMP-14 were associated with increased collagen degradation. TNF-α increased secretion of MMP-1 (p<0.01) but had no impact on MMP-1 quantities in the tissue. Immunohistochemical analysis confirmed similar tissue MMP-1 expression with or without TNF-α with epidermis being the major source of MMP-1. Increased tissue-derived collagenolytic activity with TNF-α exposure was blocked by neutralizing MMP-1 monoclonal antibody and was not due to down-regulation of tissue inhibitor of metalloproteinase-1. TNF-α increased production (p<0.01), tissue levels (p<0.005) and catalytic activity of the endogenous MMP-1 activator MMP-3. Type I collagen degradation correlated with MMP-3 tissue levels (rs=0.68, p<0.05) and was attenuated with selective MMP-3 inhibitor. Type I collagen formation was down-regulated in cultured compared with native skin explants but was not reduced further by TNF-α. TNF-α had no significant effect on epidermal apoptosis. Our data indicate that TNF-α augments collagenolytic activity of MMP-1, possibly through up-regulation of MMP-3 leading to gradual loss of type I collagen in human skin.

Successful nucleofection of rat adipose-derived stroma cells with Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe).

INTRODUCTION: Adipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs. METHODS: ASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni's post hoc test. RESULTS: High initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration. CONCLUSIONS: Our study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.

Biomechanics and biocompatibility of woven spider silk meshes during remodeling in a rodent fascia replacement model.

OBJECTIVE: The aim of this study was to investigate biomechanical and immunogenic properties of spider silk meshes implanted as fascia replacement in a rat in vivo model. BACKGROUND: Meshes for hernia repair require optimal characteristics with regard to strength, elasticity, and cytocompatibility. Spider silk as a biomaterial with outstanding mechanical properties is potentially suitable for this application. METHODS: Commercially available meshes used for hernia repair (Surgisis and Ultrapro) were compared with handwoven meshes manufactured from native dragline silk of Nephila spp. All meshes were tied onto the paravertebral fascia, whereas sham-operated rats were sutured without mesh implantation. After 4 or 14 days, 4 weeks, and 4 or 8 months, tissue samples were analyzed concerning inflammation and biointegration both by histological and biochemical methods and by biomechanical stability tests. RESULTS: Histological sections revealed rapid cell migration into the spider silk meshes with increased numbers of giant cells compared with controls with initial decomposition of silk fibers after 4 weeks. Four months postoperatively, spider silk was completely degraded with the formation of a stable scar verified by constant tensile strength values. Surgisis elicited excessive stability loss from day 4 to day 14 (P < 0.001), with distinct inflammatory reaction demonstrated by lymphocyte and neutrophil invasion. Ultrapro also showed decreasing strength and poor elongation behavior, whereas spider silk samples had the highest relative elongation (P < 0.05). CONCLUSIONS: Hand-manufactured spider silk meshes with good biocompatibility and beneficial mechanical properties seem superior to standard biological and synthetic meshes, implying an innovative alternative to currently used meshes for hernia repair.

Complete horizontal skin cell resurfacing and delayed vertical cell infiltration into porcine reconstructive tissue matrix compared to bovine collagen matrix and human dermis.

BACKGROUND: Xenogenous dermal matrices are used for hernia repair and breast reconstruction. Full-thickness skin replacement is needed after burn or degloving injuries with exposure of tendons or bones. The authors used a human skin organ culture model to study whether porcine reconstructive tissue matrix (Strattice) is effective as a dermal tissue replacement. METHODS: Skin cells or split-thickness skin grafts were seeded onto human deepidermized dermis, Strattice, and Matriderm. Cellular resurfacing and matrix infiltration were monitored by live fluorescence imaging, histology, and electron microscopy. Proliferation, apoptosis, cell differentiation, and adhesion were analyzed by immunohistochemistry. RESULTS: Epithelial resurfacing and vertical proliferation were reduced and delayed with both bioartificial matrices compared with deepidermized dermis; however, no differences in apoptosis, cell differentiation, or basement membrane formation were found. Vertical penetration was greatest on Matriderm, whereas no matrix infiltration was found on Strattice in the first 12 days. Uncompromised horizontal resurfacing was greatest with Strattice but was absent with Matriderm. Strattice showed no stimulatory effect on cellular inflammation. CONCLUSIONS: Matrix texture and surface properties governed cellular performance on tissues. Although dense dermal compaction delayed vertical cellular ingrowth for Strattice, it allowed uncompromised horizontal resurfacing. Dense dermal compaction may slow matrix decomposition and result in prolonged biomechanical stability of the graft. Reconstructive surgeons should choose the adequate matrix substitute depending on biomechanical requirements at the recipient site. Strattice may be suitable as a dermal replacement at recipient sites with high mechanical load requirements.

Matrix metalloproteinase inhibition delays wound healing and blocks the latent transforming growth factor-beta1-promoted myofibroblast formation and function.

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad-spectrum MMP inhibitor BB-94 has when applied topically to full-thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming-growth factor-beta1 (TGF-beta1). BB-94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB-94 dramatically increased the level of latent and active MMP-9. The increased levels of active MMP-9 may eventually overcome the ability of BB-94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB-94 was also found to inhibit the ability of latent TGF-beta1 to promote the formation and function of myofibroblasts. These results suggest that BB-94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte-fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF-beta1.

Magnetic resonance imaging monitoring of peripheral nerve regeneration following neurotmesis at 4.7 Tesla.

BACKGROUND: The preoperative diagnostic imaging of peripheral nerve lesions and the postoperative monitoring of microsurgically coapted nerves remain unsolved problems. The aim of this study was to investigate peripheral nerve regeneration after complete neurotmesis with magnetic resonance imaging techniques. METHODS: Study groups included 70 rats. Their right sciatic nerve was either cut and left untreated or epineurially coapted. After postoperative days 3, 6, 10, and 14 and then weekly until postoperative day 84, these rats underwent scanning at 4.7 T. T2 signal intensities of the nerves were analyzed. In parallel, on postoperative days 3, 6, 10, 14, 21, 28, 42, 63, or 84, rats were killed for histologic processing. These findings were related to the corresponding images. RESULTS: After an initial T2 signal increase of the nerves in both groups, the coapted group demonstrated a major T2 signal decrease in the distal part of the nerve after postoperative day 21, whereas in the unrepaired group a signal decrease was not observed until postoperative day 42. Differences between the two groups were significant at postoperative days 3, 6, and 28 and thereafter. The signal decrease in the coapted nerves could be correlated to the ingrowth of regenerating axons observed by histology. Moreover, the continuity of coapted nerves or an explicit gap in the unrepaired group was detectable at every time point. CONCLUSIONS: This study presents novel magnetic resonance imaging data regarding regeneration after neurotmesis. High-field-strength magnetic resonance imaging has the potential to diagnose a discontinuity within a nerve of interest and monitor its regeneration after coaptation.

Separation-induced receptor changes in the hippocampus and amygdala of Octodon degus: influence of maternal vocalizations.

Relatively little is known about the basic mechanisms that play a role in the vulnerability of the developing brain toward adverse environmental influences. Our study in the South American rodent Octodon degus revealed that repeated brief separation from the parents and exposure to an unfamiliar environment induces in the hippocampal formation of male and female pups an upregulation of D1 and 5-HT1A receptor density in the stratum radiatum and stratum lacunosum moleculare of the CA1 region. In the CA3 region, only the 5-HT1A receptors were upregulated; no changes were observed for D1 receptors in this region. GABA(A) receptor density in the hippocampus and amygdala was downregulated (nonsignificant trend) after parental separation. The acoustic presence of the mother during parental separation suppressed the D1 and 5-HT1A receptor upregulation in some regions of the hippocampus; no such suppressing influence was observed for the GABA(A) receptors. In the basomedial amygdala, the maternal calls enhanced the separation-induced 5-HT1A receptor upregulation in the male pups, whereas in the female pups the separation-induced receptor densities were not only suppressed by the maternal call but further downregulated, compared with the control group. These results demonstrate that early adverse emotional experience alters aminergic function within the hippocampus and amygdala and that the mother's voice, a powerful emotional signal, can modulate these effects in the developing limbic system.