Current Tissue Engineering (Discontinued) - Volume 5, Issue 2, 2016

Collagens are a key element in the architecture of all organs and tissues. These proteins not only build the extracellular scaffolds that define the mechanical properties of tissues, but also play an important role as cell signaling molecules. Certain characteristics of collagens enable them to fulfill specific functions, including their triple-helical structure and their ability to self-assemble into complex extracellular structures. Their unique properties allow collagens to serve as a material to build scaffolds for tissue repair and engineering, as a drug delivery vehicle, and, in the form of gelatin, as a gelling agent in food, pharmaceutical, and cosmetic industries. Animal-derived collagens are widely utilized in the biomedical field today, but their use is associated with a number of limitations and potential side effects. Efforts over the last two decades have advanced technology for the production of recombinant variants of human collagens and collagen-like proteins. Potential applications of these proteins not only eliminate the risks associated with animal-derived collagens, but also offer customized qualities of rationally designed collagen-like proteins. This review highlights the current state of the development of the recombinant collagen technology. Moreover, it discusses key physicochemical and biological parameters that define the collagenous nature of novel recombinant collagen variants.

The research for regenerative medicine has currently focused on the development of pluripotent cells, i.e., embryonic stem cells and induced pluripotent cells. These cells have been proven to differentiate target cells in vitro, but they could not reproduce an organized arrangement with other types of cells and extracellular matrices, including collagens, elastins, proteoglycans, and others. Although growth factors influence cells to proliferate and differentiate target cells, most of them are unstable or diffusible in vivo. Growth factors, designed to bind to specific extracellular matrices, have been introduced to the tissue regeneration. Fabrication and development of three-dimensional structures are highly desired to regenerate tissues and organs large enough for transplantation. Collagen is the major extracellular matrix in mammals, also found in the animals belonging to the phylum polifera, e.g., sponges, and distributed in the jelly-like mesophyl between two thin cell layers. Therefore, collagen is the oldest extracellular matrix providing a scaffold for cells in multicellular organisms. Collagen is a protein family consisting of 28 different types, which polymerize into fibrils or basement membranes. By fabricating graded structures specific for target tissues and organs, one can obtain suitable scaffolds for tissue regeneration. Decellularized scaffolds would presently be one of the best options because they can maintain the basic architecture of extracellular matrices such as tissue size. In this review, the origin, polymerized structure, and graded arrangement of collagen in extracellular space will be discussed. Some examples of a bioreactor to regenerate the tissue constructs together with collagen and cells are also presented.

Human skin equivalents (HSEs), which are three-dimensional (3D) organotypic culture models, are essential tools to examine a wide range of hypotheses on the structure and function of epithelial tissues, structural assembly of extracellular matrix, and dermal-epidermal interactions. Here, we review epidermal basement membrane formation in HSEs focusing on enhancers of basement membrane formation (especially inhibitors of extracellular matrix-degrading enzymes) with potential applications in skin biology research and tissue engineering. Exogenous addition of laminin-332 or type IV collagen in HSEs increases expression and deposition of types IV and VII collagen and enhances epidermal basement membrane assembly in HSEs. These results suggested that epidermal basement membrane structure would also be enhanced by inhibition of enzymes that degrade extracellular matrix components produced by the cells. Indeed, epidermal basement membrane components produced by keratinocytes and fibroblasts in HSEs are concentrated and stabilized at the dermal-epidermal interface in the presence of synthetic inhibitors of matrix metalloproteinases, plasmin, and heparanase. Increased local concentrations of epidermal basement membrane components may provide a favorable microenvironment at the dermalepidermal junction for formation of epidermal basement membrane structures, including lamina lucida, lamina densa, lamina fibroreticularis and anchoring complex. Inhibitors of extracellular matrixdegrading enzymes therefore allow us to generate more stable 3D culture models for laboratory investigations of the regulatory mechanisms of in vivo skin structure and function, and may also prove valuable for improving the clinical outcome after grafting of skin substitutes.

Recent research suggests that the basement membrane at the dermalepidermal junction of the skin plays an important role in maintaining a healthy epidermis and dermis, and repeated damage to the skin can destabilize the skin and accelerate the aging process. Skin-equivalent models are suitable for studying the reconstruction of the basement membrane and its contribution to epidermal homeostasis because they lack the basement membrane and show abnormal expression of epidermal differentiation markers. By using these models, it has been shown that reconstruction of the basement membrane is enhanced not only by supplying basement membrane components, but also by inhibiting proteinases such as urokinase and matrix metalloproteinase. Although matrix metalloproteinase inhibitors assist in the reconstruction of the basement membrane structure, their action is not sufficient to promote its functional recovery. However, heparanase inhibitors stabilize the heparan sulfate chains of perlecan (a heparan sulfate proteoglycan) and promote the regulation of heparan sulfate binding growth factors in the basement membrane. Heparan sulfate promotes effective protein-protein interactions, thereby facilitating the assembly of type VII collagen anchoring fibrils and elastin-associated microfibrils. Using both matrix metalloproteinase inhibitors and heparanase inhibitors, the basement membrane in a skinequivalent model comes close to recapitulating the structure and function of an in vivo basement membrane. Therefore, by using an appropriate dermis model and suitable protease inhibitors, it may be possible to produce skin-equivalent models that are more similar to natural skin.

Purpose: To compare the biosafety of chitosan (CHM) and collagen– vitrigel biomembranes (CVM) when implanted to the anterior chamber of an animal model to set an optimal scaffold for further corneal engineering research. Methods: Four White New Zealand rabbits, 3 months old, were implanted with CHM in one eye, and other four rabbits were implanted with CVM membranes following cold burn damage on the corneal surface. The contralateral eye was used as the control. After 1 week, rabbits were sacrificed, and the obtained corneas were clinically evaluated and processed for histological analysis. Results: Eyes implanted with CHM developed severe inflammation with 360° neovascularization, ciliary injection, optical opacity, and purulent exudate in the anterior chamber. Microscopically, CHM-implanted eyes showed severe exudative, inflammatory, and necrotic processes that were mainly composed of polymorphonuclear (PMN) leukocytes, cellular debris, and macrophages. Eyes implanted with CVM showed little or no signs of clinical inflammation. Histological analysis of the CVM and control eyes showed no signs of inflammation, except in places where corneal suture ports and closure with a suture were performed. Conclusions: CHM are not biocompatible for ocular purposes. CVM are safe to be used for further in vivo research as cell scaffold in corneal engineering.

Background: Demineralized bone matrix is used clinically to stimulate bone repair in orthopedics and dentistry. Demineralized bone matrix has osteoconductive and osteoinductive properties that contribute to its efficacy in new bone formation. However, ethylene-oxide sterilization in the clinical setting diminishes this efficacy. Studies using intramuscular implantation of demineralized bone matrix in rats demonstrate deterioration of its osteoconductive properties by ethylene oxide sterilization. Objective: The goal of this study is to investigate whether treatment with chitosan which is osteoconductive will improve the osteoconductivity of ethylene-oxide sterilized demineralized bone matrix and thus restore the original osteogenetic efficacy of demineralized bone matrix. Methods: We implanted normal and modified demineralized bone matrix implants into the abdominal muscles of Sprague-Dawley rats. New bone growth in implants, harvested at 4 weeks, was determined by mineral content, bone alkaline phosphatase activity, and histology. Results: The unmodified demineralized bone matrix implants demonstrated extensive areas of trabecular bone containing osteoblasts and osteocytes. Ethylene-oxide sterilization of demineralized bone matrix resulted in fibrosis, rather than new bone formation, in the intramuscular implantation site in the rat. Treatment of ethylene-oxide sterilized demineralized bone matrix with chitosan restored mineral content and bone alkaline phosphatase activity of these samples to control levels. Conclusion: Treatment of ethylene-oxide sterilized demineralized bone matrix with chitosan restores the osteoconductive properties completely so that new bone formation is comparable to that of nonsterilized demineralized bone matrix. Thus chitosan treatment of ethylene-oxide sterilized demineralized bone matrix may be used to restore the clinical efficacy of demineralized bone matrix after sterilization.

Background: Currently, various approaches employed for treating diabetic wounds face several limitations. Since diabetic ischemia-related non-healing wounds cause economic burden and social problems world-wide, newer methods need to be developed to address the crisis. Objective: In this study, we tested a novel strategy of applying hybrid scaffold developed from synthetic biodegradable electro-spun poly[Lactide-glycolidecaprolactone] and bio-mimetic fibrin based matrix combined with autologous circulating progenitor cells on wounds in diabetic rabbits. Methods: The wounds created in rabbit ear were grouped into three categories: (i) untreated open wounds [control]; (ii) covered with hybrid scaffold [test1]; and (iii) applied with autologous progenitor cell suspension and covered with hybrid scaffold [test 2]. Replicate wounds were explanted at 3 time periods ending 28 days, gross tissue and histological sections were compared between control and tests. Healing parameters assessed were collagen organization, angiogenesis and epithelial coverage. Survival of transplanted cells at the wound site was tracked. Results: All wounds healed by 28 days; but, fastest epithelial healing and least scar formations were achieved when wounds were applied with progenitors and scaffold together. Collagen organization and angiogenesis were the best in Test2 followed by Test1 and was minimal in Control as compared to normal skin. Upon cell transplantation, healed skin thickness was near normal with appendage-like structures. Conclusion: Transplanted cells could be tracked till the end of the study through fluorescence imaging. The transplanted cells seemed important for dermal and epidermal regeneration. Even though a mixture of cells was transplanted, all of them can be harvested easily from autologous source and could be committed to respective lineage within few days. Therefore, it could be a potential strategy for regeneration of wounds in human subjects.