Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.
Articular cartilage degeneration and traumatic cartilage defects are common disorders causing pain and disability. Many strategies were developed throughout years to address these problems. Tissue engineering approaches offer several promising solutions. Articular hyaline cartilage has very complex structure and follows a multi-layer architecture. In this study, we developed a 4-layers scaffold mimicking the fibers orientation observed within the cartilage extra-cellular matrix (ECM). Bone and cartilage microparticles were incorporated in the first and second layers, respectively, to direct the differentiation of stem cells toward subchondral bone and cartilage phenotype. The 2 upper layers were designed with random (layer 3) and aligned (layer 4) electrospun nanofibers in an attempt to mimic hyaline articular cartilage ECM orientation. Histological analysis of the scaffold showed a continuous and homogeneous structure. Moreover, we confirmed the presence of cartilage and bone microparticles in the gelatin foam-based layers (layer 1 and 2). Scanning electron microscope (SEM) confirmed the tubular, vertical, morphology of the first and second layers, thereby mimicking as intended the native hyaline cartilage structure. Water uptake and degradation rate of our scaffold appeared suitable for our application, with many options for tunability. Finally, we demonstrated that the scaffolds were biocompatible, as it promoted the survival and attachment of MIAMI cells in vitro. In this study, we described the first phases of the development of a multi-layered scaffold designed to mimic the native architecture of articular hyaline cartilage. With further refinements and testing, we hope that this strategy will pave the way for potential new advances in the field of osteochondral repair. Keywords: Electrospinning, gelatin, human tissues, hyaline cartilage, MIAMI cells, multi-layered scaffold, nanofibers, tissue engineering.
Regenerative medicine (RM) possesses the ability to repair injured as well as aging cells, tissues and organs. This review discusses the recent advances, future prospects and challenges in the field of RM mainly the stem cell therapy in degenerative retinal diseases such as age related macular degeneration (AMD), retinitis pigmentosa (RP) and Stargardt disease (STGD). Stem cell lines like retinal progenitor cells, induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells and very small embryonic-like stem cells have potential application in the degenerative retinal diseases where they have the potency to regenerate the injured and damaged photoreceptors and retinal neurons ultimately restoring and enhancing the visual functions. Stem cell transplantation for AMD, RP and STGD is currently transforming from bench side research to bedside which may eventually allow the progress of the more advanced therapies. In future, stem cell therapy could be combined with bioengineered products and small molecules to produce novel therapies in the field of ophthalmology particularly in terms of visual recovery to fight against blindness. Keywords: Age-related macular degeneration, Retinitis Pigmentosa, Regenerative Medicine, Stargardt disease, Stem Cells.
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. Keywords: Bone alkaline phosphatase, bone morphogenetic protein, chitosan, demineralized bone matrix, ethyleneoxide sterilization, mineralization, osteoconductive, osteoinductive.
Retinal degeneration due to injury or disease can be a devastating event to the quality of life of an individual. Efforts to develop strategies for retinal repair and regeneration have been challenging due to the complexities in the microenvironment necessary to support and maintain retinal function, as well as the difficulty in guiding the correct integration of regenerating cells to the existing neural networks. Stem cell therapies have recently emerged as a viable option to promote retinal repair and regeneration with encouraging results. As more studies are performed on the effect of stem cells on the regenerative processes in the retina, it becomes clear that stem cells can play a beneficial role not only by their differentiation capabilities and cellular replacement potential, but also as modulators of local environmental factors to provide conditions that are more conducive to functional repair and regeneration. Stem cells have been shown to be able to modulate several processes that are detrimental to regeneration such as inflammation, extracellular matrix remodeling, and loss of trophic support, among others. Here we review some of the latest studies on stem cell-mediated retinal regeneration by focusing primarily on how stem cells can modulate the microenvironment in the neural retina to augment its regenerative potential following injury or disease. Keywords: Cell therapy, microenvironmental modulation, neuroprotection, retinal regeneration, regenerative medicine, regeneration, stem cells.
Structural allograft bone transplantation has evolved over many years and despite improvements in fixation and advances in graft safety, there are significant limitations and potential complications. This has prompted investigations in our laboratory to reduce bone to its smallest common denominator and to combine the inductive potential of small particle allograft bone with somatic stem cells and inorganic matrices as a regenerative engineering strategy for skeletal reconstruction. Preliminary animal studies identified an optimal particle size that since has been corroborated in human trials for closed segment bone defects. Further studies have confirmed the invigorating effects of stem cells cultured with bone particles that too have been observed and confirmed in human transplantation. The addition of structural inorganic materials to particulate bone and cells presents an alternative to large structural allografts. Newer technologies that alter the surface properties of inorganic and organic matrices offer unique biologic and physical pathways to bone induction that may be transformative in the field of bone regeneration and provide superior clinical alternatives to segmental bone loss. Keywords: Allograft, bone regeneration, mimetic material, PEEK, stem cells, transplantation.
Retinal ganglion cell (RGC) degeneration is one of the major causes for blindness, a condition widely attributed to the pathology of RGC axons. How to promote the affected RGC axons to regenerate is currently under intense investigation. Difficulties in adult RGC axons to regenerate are due partially to the attenuated intrinsic re-growth ability, and further complicated by factors located in the unfavorable microenvironments, especially the myelin sheath and the activated glia cells. Additionally, axon damage often induces RGC death, eliminating any chance for axon regeneration, and therefore further complicates the treatment for RGC degeneration. In this review, we discuss the different aspects that cause regeneration failure in the RGC axons, and also the currently known factors that promote RGC axon regeneration ability. These findings are encouraging and open the possibility that clinically meaningful regeneration may become achievable one day in the future. Keywords: Axon damage, axon regeneration, cell replacement, retinal ganglion cell (RGC).
Background: Age-related macular degeneration (AMD) is a progressive degenerative disease of the macula and is the leading cause of visual impairment in the elderly population worldwide. The two advanced forms of AMD are geographic atrophy and choroidal neovascularization (CNV) which are the major causes of visual loss in AMD patients. Clinically effective therapy for CNV is now available but is limited by the need for repeated intravitreal injections of anti-angiogenic agents. There is still no effective treatment for geographic atrophy. Earlier concepts on AMD therapy were confined to local ocular therapies. However, recent evidences show that the complement system, inflammatory response and oxidative stress level are altered in the systemic circulation of AMD patients. Meanwhile, within regenerative medicine, stem cell therapy is effectively alleviating these systemic changes in multiple diseases. These findings have provided new insight into the pathophysiology and subsequent potential for clinical interventions in AMD. Methods: This review article will first summarize the pathophysiology of AMD and then describe the systemic disorders associated with AMD. In addition, the characteristics of mesenchymal stem cells (MSCs) in regenerative medicine and its potential therapeutic effect for systemic disorders will also be discussed. Results: AMD is an oxidative stress and inflammation-related disease affecting the RPE and photoreceptor cells of the macula. It is not only a localized eye disease, but also a disease affected by the systemic status of the patient. There is good evidence to show the influence of systemic factors in the pathogenesis, including complement system, inflammation, oxidative stress and angiogenic factors. Since MSCs possess the paracrine effects of anti-oxidation, anti-inflammation and immunomodulation, systemic administration of MSCs could theoretically alter the systemic influence on AMD progression. Currently, there are 3 MSC clinical trials on AMD, which locally apply bone marrow and adipose tissue-derived MSCs to supply neurotrophic factors to the microenvironment. Conclusion: MSCs hold promises for treating the systemic conditions associated with AMD. The anti-inflammatory and anti-oxidative effects of MSCs are desirable and be developed as a potential therapeutic strategy against AMD for clinical treatment in the future. Keywords: Age-related macular degeneration, complement system, inflammation, mesenchymal stem cells, oxidative stress.
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. Keywords: Autologous cell transplantation, bio mimetic scaffolds, circulating progenitors, diabetic wound regeneration.
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. Keywords: 3D-culture, Epidermal basement membrane, heparanase, human skin equivalents, matrix metalloproteinase, plasmin, synthetic inhibitor.
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. Keywords: Collagen, connective tissue, gelatin, recombinant collagen, tissue engineering, tissue regeneration.
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. Keywords: Chitosan, collagen-vitrigel, cornea, corneal endothelium, rabbit, tissue scaffold.
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. Keywords: 3D scaffold, acellularized extracellular matrix, basement membrane, collagen fibril, extracellular matrix, graded structure, growth factors.
