Skin protects the body from exogenous substances and functions as a barrier to fluid loss and trauma. The skin comprises of epidermal, dermal and hypodermal layers, which mainly contain keratinocytes, fibroblasts and adipocytes, respectively, typically embedded on extracellular matrix made up of glycosaminoglycans and fibrous proteins. When the integrity of skin is compromised due to injury as in burns the coverage of skin has to be restored to facilitate repair and regeneration. Skin substitutes are preferred for wound coverage when the loss of skin is extensive especially in the case of second or third degree burns. Different kinds of skin substitutes with different features are commercially available; they can be classified into acellular skin substitutes, those with cultured epidermal cells and no dermal components, those with only dermal components, and tissue engineered substitutes that contain both epidermal and dermal components. Typically, adult wounds heal by fibrosis. Most organs are affected by fibrosis, with chronic fibrotic diseases estimated to be a leading cause of morbidity and mortality. In the skin, fibroproliferative disorders such as hypertrophic scars and keloid formation cause cosmetic and functional problems. Dermal fibroblasts are understood to be heterogeneous; this may have implications on post-burn wound healing since studies have shown that superficial and deep dermal fibroblasts are anti-fibrotic and pro-fibrotic, respectively. Selective use of superficial dermal fibroblasts rather than the conventional heterogeneous dermal fibroblasts may prove beneficial for post-burn wound healing.

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Bioengineered artificial blood vessels have been a major area of interest over the last decade. Of particular interest are small diameter vessels, as surgical options are currently limited. This study aimed to fabricate a small diameter, heterogeneous bilayer blood vessel-like construct in a single step with gelatin methacryloyl (GelMA) bioink using a 3D micro-extrusion bioprinter on a solid platform. GelMA was supplemented with Hyaluronic acid (HA), glycerol and gelatin to form a GelMA bioink with good printability, mechanical strength, and biocompatibility. Two separate concentrations of GelMA bioink with unique pore sizes were selected to fabricate a heterogeneous bilayer. A higher concentration of GelMA bioink (6% w/v GelMA, 2% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load human umbilical vein endothelial cells (HUVECs) and form an inner, endothelial tissue layer. A lower concentration of GelMA bioink (4% w/v GelMA, 4% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load smooth muscle cells (SMCs) and form an outer, muscular tissue layer. Bioprinted blood vessel-like grafts were then assessed for mechanical properties with Instron mechanical testing, and suture-ability, and for biological properties including viability, proliferation, and histological analysis. The resulting 20 mm long, 4.0 mm diameter lumen heterogeneous bilayer blood vessel-like construct closely mimics a native blood vessel and maintains high cell viability and proliferation. Our results represent a novel strategy for small diameter blood vessel biofabrication.

Burns are a significant cause of trauma, and over the years, the focus of patient care has shifted from just survival to facilitation of improved functional outcomes. Typically, burn treatment, especially in the case of extensive burn injuries, involves surgical excision of injured skin and reconstruction of the burn injury with the aid of skin substitutes. Conventional skin substitutes do not contain all skin cell types and do not facilitate recapitulation of native skin physiology. Three-dimensional (3D) bioprinting for reconstruction of burn injuries involves layer-by-layer deposition of cells along with scaffolding materials over the injured areas. Skin bioprinting can be done either in situ or in vitro. Both these approaches are similar except for the site of printing and tissue maturation. There are technological and regulatory challenges that need to be overcome for clinical translation of bioprinted skin for burn reconstruction. However, the use of bioprinting for skin reconstruction following burns is promising; bioprinting will enable accurate placement of cell types and precise and reproducible fabrication of constructs to replace the injured or damaged sites. Overall, 3D bioprinting is a very transformative technology, and its use for wound reconstruction will lead to a paradigm shift in patient outcomes. In this review, we aim to introduce bioprinting, the different stages involved, in vitro and in vivo skin bioprinting, and the various clinical and regulatory challenges in adoption of this technology.

Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules that regulate collagen fibrillogenesis and inhibit transforming growth factor-β activity; thus, they may play a critical role in wound healing and scar formation. Hypertrophic scarring is a dermal form of fibroproliferative disorders, which occurs in over 70% of burn patients and leads to disfigurement and limitations in function. By understanding the cellular and molecular mechanisms that lead to scarring after injury, new clinical therapeutic approaches can by developed to minimize abnormal scar formation in hypertrophic scarring and other fibroproliferative disorders. To study the expression and localization of SLRPs with connective tissue cells in tissue immunohistochemistry, immunofluorescence staining, immunoblotting, and reverse-transcription polymerase chain reaction were used in normal skin and hypertrophic scar (HTS). In normal skin, there was more decorin and fibromodulin accumulation in the superficial layers than in the deeper dermal layers. The levels of decorin and fibromodulin were significantly lower in HTS, whereas biglycan was increased when compared with normal skin. There was an increased expression of biglycan, fibromodulin, and lumican in the basement membrane and around basal epithelial cells. In contrast, these proteoglycans were absent or weakly expressed in HTS. The findings suggest that down-regulation of SLRPs after wound healing in deep injuries to the skin plays an important role in the development of fibrosis and HTS.

Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders. Fibroblasts from the deep dermis are implicated in the development of HTS after injuries that involve deeper areas of the skin. However, fibroblasts that reside in the superficial layer of the skin show antifibrotic properties, and injuries limited to this area heal with little or no scarring. Previously, cellular and molecular characteristics of superficial fibroblasts and deep dermal fibroblasts that may influence HTS formation were analyzed. In this study, differences in cellular behavior between superficial fibroblasts and deep dermal fibroblasts that may also affect the development of HTS or tissue fibrosis were further characterized. Immunostaining and migration, adhesion, apoptosis, and cell viability assays were performed in fibroblasts from the superficial and deep dermis. Reverse-transcription polymerase chain reaction was used to examine the gene expression of molecules involved in cell death after treatment of fibroblasts with decorin. When compared with superficial fibroblasts, deep dermal fibroblasts showed lower migration rates. Although all the fibroblasts tested showed no difference in adhesion to fibronectin, superficial fibroblasts demonstrated increased apoptotic and dead cells when treated with decorin. Decorin resulted in a significant increase in the expression of apoptosis markers, histone-1, caspase-1, caspase-8, and p53 in superficial fibroblasts when compared with deep dermal fibroblasts. Taken together, the findings suggest that reduced migration, lack of decorin, and resistance of deep dermal fibroblasts to decorin-induced apoptosis may result in hypercellularity in injuries involving the deep dermis, leading to deposition of excess extracellular matrix and HTS formation.

Growth factors (GFs) are endogenous proteins capable of acting on cell-surface receptors and directing cellular activities involved in the regeneration of new bone tissue. The specific actions and long-term effects of GFs on bone-forming cells have resulted in exploration of their potential for clinical bone repair. The concerted efforts have led to the recent approval of two GFs, bone morphogenetic protein-2 and osteogenic protein-1, for clinical bone repair, and human parathryroid hormone (1-34) for augmentation of systemic bone mass. This review provides a selective summary of recent (2001-2004) attempts for GF delivery in bone tissue regeneration. First, a summary of non-human primate studies involving local regeneration and repair is provided, with special emphasis on the range of biomaterials used for GF delivery. Next, efforts to administer GFs for systemic augmentation of bone tissue are summarised. Finally, an alternative means of GF delivery, namely the delivery of genes coding for osteogenic proteins, rather than the delivery of the proteins, is summarised from rodent models. To conclude, future avenues of research considered promising to enhance the clinical application of GFs are discussed.

Hypertrophic scar (HTS) occurs after injuries involving the deep dermis, while superficial wounds (SWs) to the skin heal with minimal or no scarring. The levels of transforming growth factor (TGF)-β1 and small leucine-rich proteoglycans (SLRPs) with fibroblast subtype and function may influence the development of HTS. The aim of this study was to characterize the expression and localization of factors that regulate wound healing including SLRPs, TGF-β1, and TGF-β3 in an experimental human SW and deep wound (DW) scar model including fibroblasts from superficial and deep layers of normal dermis. A 6-cm horizontal dermal scratch experimental wound was created, which consisted of progressively deeper wounds that were superficial at one end (0-0.75 mm deep) and deep (0.75-3 mm deep) at the other end, located on the anterior thigh of an adult male. Immunofluorescence staining, immunoblotting, reverse transcription polymerase chain reaction, and flow cytometry were performed to analyze the cellular and molecular differences between the SW scar and DW scar as well as fibroblasts isolated from superficial layer (L1) and deep layer (L5) of normal dermis. Comparing SWs and L1 fibroblasts, the expression of decorin, fibromodulin, and TGF-β3 was considerably lower than in DWs and L5 fibroblasts; however, TGF-β1 was higher in the deeper dermal wounds. When compared with L1 fibroblasts, L5 fibroblasts had lower Thy-1 immunoreactivity and significantly higher expression of TGF-β receptor type II. Decreased antifibrotic molecules in matrix of deep dermis of the skin and the unique features of the associated fibroblasts including an increased sensitivity to TGF-β1 stimulation contribute to the development of HTS after injuries involving the deep dermis.