Hybrid scaffolds for cartilage tissue engineering provide the potential for high stiffness properties in tension and compression while exhibiting the viscoelastic response found in hydrogels and native cartilage tissue. We investigate the impact of a hybrid scaffold fabricated from a hyaluronic acid (HA)-based hydrogel combined with porous poly(ε-caprolactone) (PCL) material formed by a particulate leaching method to study dedifferentiated chondrocyte response. The material properties of the hybrid scaffold showed mean Young's moduli in tension which were similar to human articular cartilage but not statistically different between the hybrid and porous PCL scaffolds at 2.02 and 2.07 MPa, respectively. For both the hybrid and porous PCL control scaffolds in compression at low loading frequencies (<1 Hz) and 10% strain peak amplitude the Young's moduli are not statistically distinct. However, at frequencies in the range of normal human gait from 1 to2 Hz, hybrid scaffolds exhibit significantly (p < 0.01) increased loss moduli indicating additional contribution of the viscous phase to stiffness. Dedifferentiated chondrocytes seeded onto the scaffolds exhibited a rounded morphology in hybrid scaffolds however ECM protein expression levels of collagen type I, collagen type II, and aggrecan are not different from the PCL control scaffolds. These results provide a model platform to investigate cell response to mechanical and chemical cues in a hybrid scaffold system with mechanical properties similar to human cartilage that does not contribute to differentiation in order to identify the appropriate design and development parameters to promote formation of extracellular matrix and investigate chondrocyte scaffold interactions.

Tyramine-substituted hyaluronic acid (HA-Tyr) hydrogels formed by the oxidative coupling reaction of hydrogen peroxide (HO) and horseradish peroxidase (HRP) have been used for cellular encapsulation and protein delivery. Crosslinking density and gelation time can be tuned by altering the HO and HRP concentrations. Previous studies using HA-Tyr constructs report significant mechanical degradation after 21 days of culture. In this work, exogenous supplementation of HRP after initial gelation resulted in superior mechanical properties in acellular hydrogels and improved viability and proliferation in cell-laden constructs. Swelling of the acellular hydrogels was prevented in the samples receiving exogenous HRP. Monolayer studies showed no negative effect of relevant HRP concentrations on the viability of human adipose-derived stem cells (hASCs) and improved the viability of hASCs cultured with HRP and HO compared to HO alone. Taken together, this study demonstrates that HA-Tyr hydrogel properties could be modified by exogenous supplementation of HRP to tune scaffold degradation and improve cell viability by mitigating the negative effects of oxidative stress.

Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Prior studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, while UVA induces LAGT secondary to reactive oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage, and determine an optimal wavelength to maximize safety and efficacy. Results: UV at 288, 311 and 320nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4kJ/m2 at 311nm. Consistent with its robust cytotoxicity, 288nm produced significantly high levels of DNA damage at all doses tested, while 311, 320 and 330nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. While 288nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ/m2 at 311nm. An in vivo pilot study assessing 311nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (p<0.05) increase in transduction with insignificant cytotoxicity. Conclusion: 311nm was found to be the optimal wavelength for LAGT based on its superior efficacy at the peak dose, and its broad safety range that is remarkably wider than the other UV wavelengths tested.

Objective: The purpose of this study was to investigate whether low-level light therapy (LLLT) was capable of modulating expression of ultraviolet (UV) light-responsive genes in vivo. Materials and methods: The effects of 670 nm light-emitting diode (LED) array irradiation were investigated in a hairless SHK-1 mouse epidermis model. Mice were given a single dose of UVA/UVB light, or three doses of red light (670 nm @ 8 mW/cm 2 x 312 sec, 2.5 J/cm 2 per session) spread over 24 h along with combinations of pre-and post-UV treatment with red light. Levels of 14 UV-responsive mRNAs were quantified 24 h after UV irradiation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: The transcription of mRNAs encoding for cluster of differentiation molecule 11b (CD11b) ( p < 0.05) and interferon (IFN)-c ( p < 0.012) increased after irradiation with red light alone, whereas expression level of cyclooxygenase (COX)-2 ( p < 0.02) was downregulated. Genes unresponsive to UV did not change their expression levels after exposure to red light either. Pretreatment with red light significantly modified response of Fos to UV exposure ( p < 0.01). A synergy of UV and post-treatment with red light in reducing the transcription levels of CD11b ( p < 0.05) and inducible nitric oxide synthase (iNOS) ( p < 0.05) was observed. Conclusions: This is an initial observation that in mouse red light LLLT more often than not causes opposite gene expression changes or reduces those caused by moderate UVA-UVB irradiation.

Clinical application of skin substitute is typically a two‐stage procedure with application of skin substitute matrix to the wound followed by engraftment of a split‐thickness skin graft (STSG). This two‐stage procedure requires multiple interventions, increasing the time until the wound is epithelialised. In this study, the feasibility of a one‐stage procedure by combining bioengineered collagen‐chondroitin‐6‐sulfate (DS1) or decellularised fetal bovine skin substitute (DS2) with autologous skin cell suspension (ASCS) in a porcine full‐thickness wound healing model was evaluated. Twelve full‐thickness excisional wounds on the backs of pigs received one of six different treatments: empty; ASCS; DS1 with or without ASCS; DS2 with or without ASCS. The ASCS was prepared using a point‐of‐care device and was seeded onto the bottom side of DS1, DS2, and empty wounds at 80 000 cells/cm2. Wound measurements and photographs were taken on days 0, 9, 14, 21, 28, 35, and 42 post‐wounding. Histological analysis was performed on samples obtained on days 9, 14, 28, and 42. Wounds in the empty group or with ASCS alone showed increased wound contraction, fibrosis, and myofibroblast density compared with other treatment groups. The addition of ASCS to DS1 or DS2 resulted in a marked increase in re‐epithelialisation of wounds at 14 days, from 15 ± 11% to 71 ± 20% (DS1 vs DS1 + ASCS) or 28 ± 14% to 77 ± 26 (DS2 vs DS2 + ASCS) despite different mechanisms of tissue regeneration employed by the DS used. These results suggest that this approach may be a viable one‐stage treatment in clinical practice.