Large-sized or deep skin wounds require skin substitutes for proper healing without scar formation. Therefore, multi-layered skin substitutes that mimic the genuine skin anatomy of multiple layers have attracted attention as suitable skin substitutes. In this study, a novel skin substitute was developed by combining the multi-layer skin tissue reconstruction method with the combination of a human-derived keratinic extract-loaded nano- and micro-fiber using electrospinning and a support structure using 3D printing. A polycaprolactone PCL/keratin electrospun scaffold showed better cell adhesion and proliferation than the keratin-free PCL scaffold, and keratinocytes and fibroblasts showed better survival, adhesion, and proliferation in the PCL/keratin electrospun nanofiber scaffold and microfiber scaffold, respectively. In a co-culture of keratinocytes and fibroblasts using a multi-layered scaffold, the two cells formed the epidermis and dermal layer on the PCL/keratin scaffold without territorial invasion. In the animal study, the PCL/keratin scaffold caused a faster regeneration of new skin without scar formation compared to the PCL scaffold. Our study showed that PCL/keratin scaffolds co-cultured with keratinocytes and fibroblasts promoted the regeneration of the epidermal and dermal layers in deep skin defects. Such finding suggests a new possibility for artificial skin production using multiple cells.

Due to the morphological resemblance between the electrospun nanofibers and extracellular matrix (ECM), electrospun fibers have been widely used to fabricate scaffolds for tissue regeneration. Relationships between scaffold morphologies and cells are cell type dependent. In this study, we sought to determine an optimum electrospun fiber diameter for human vascular smooth muscle cell (VSMC) regeneration in vascular scaffolds. Scaffolds were produced using poly(caprolactone) (PCL) electrospun fiber diameters of 0.5, 0.7, 1, 2, 2.5, 5, 7 or 10 μm, and VSMC survivals, proliferations, infiltrations, and phenotypes were recorded after culturing cells on these scaffolds for one, four, seven, or 10 days. VSMC phenotypes and macrophage infiltrations into scaffolds were evaluated by implanting scaffolds subcutaneously in a mouse for seven, 14, or 28 days. We found that human VSMC survival was not dependent on the electrospun fiber diameter. In summary, increasing fiber diameter reduced VSMC proliferation, increased VSMC infiltration and increased macrophage infiltration and activation. Our results indicate that electrospun PCL fiber diameters of 7 or 10 µm are optimum in terms of VSMC infiltration and macrophage infiltration and activation, albeit at the expense of VSMC proliferation.

Chronic osteomyelitis is mostly caused by bacteria such as S. aureus, and is often treated with oral antibiotics or injections to suppress the bacteria. In severe cases, however, surgical treatment using antibiotic beads and metal supports may be required. In these surgeries, bacterial attachment to the metal may lead to biofilm formation and reduce antibiotics’ penetration to the bacteria. Reoperation must be performed to prevent bacterial inflammatory reactions and antibiotic resistance. Thus, in this study, we developed a dual-drug-releasing PCL/sodium-alginate-based 3D-printed scaffold to effectively treat osteomyelitis by removing the biofilm. We proposed an antibiotic-loaded biodegradable polymer scaffold using 3D printing, which was encapsulated by a second antibiotic-containing hydrogel. Then, we successfully established a dual-drug-based scaffold that consisted of a cefazolin (CFZ)-containing polycaprolactone 3D scaffold and a rifampicin (RFP)-loaded alginate hydrogel encapsulating the 3D scaffold. Our scaffold showed a synergistic effect, whereby biofilm formation was inhibited by RFP, which is an external drug, and bacterial activity was inhibited by CFZ, which is an internal drug that increases antibacterial activity. We also confirmed that the dual-drug-based scaffold did not affect the proliferation of human osteoblasts. Our findings suggest that this dual drug delivery system may serve as a new therapeutic treatment for osteomyelitis that overcomes the limitations of individual drugs.

Cartilage is a connective tissue that constitutes the structure of the body and consists of chondrocytes that produce considerable collagenous extracellular matrix and plentiful ground substances, such as proteoglycan and elastin fibers. Self-repair is difficult when the cartilage is damaged because of insufficient blood supply, low cellularity, and limited progenitor cell numbers. Therefore, three-dimensional (3D) culture systems, including pellet culture, hanging droplets, liquid overlays, self-injury, and spinner culture, have attracted attention. In particular, 3D spheroid culture strategies can enhance the yield of exosome production of mesenchymal stem cells (MSCs) when compared to two-dimensional culture, and can improve cellular restorative function by enhancing the paracrine effects of MSCs. Exosomes are membrane-bound extracellular vesicles, which are intercellular communication systems that carry RNAs and proteins. Information transfer affects the phenotype of recipient cells. MSC-derived exosomes can facilitate cartilage repair by promoting chondrogenic differentiation and proliferation. In this article, we reviewed recent major advances in the application of 3D culture techniques, cartilage regeneration with stem cells using 3D spheroid culture system, the effect of exosomes on chondrogenic differentiation, and chondrogenic-specific markers related to stem cell derived exosomes. Furthermore, the utilization of MSC-derived exosomes to enhance chondrogenic differentiation for osteoarthritis is discussed. If more mechanistic studies at the molecular level are conducted, MSC-spheroid-derived exosomes will supply a better therapeutic option to improve osteoarthritis.