3D printed PLA-based scaffolds

Rapid prototyping (RP), also known as additive manufacturing (AM), has been well received and adopted in the biomedical field. The capacity of this family of techniques to fabricate customized 3D structures with complex geometries and excellent reproducibility has revolutionized implantology and regenerative medicine. In particular, nozzle-based systems allow the fabrication of high-resolution polylactic acid (PLA) structures that are of interest in regenerative medicine. These 3D structures find interesting applications in the regenerative medicine field where promising applications including biodegradable templates for tissue regeneration purposes, 3D in vitro platforms for studying cell response to different scaffolds conditions and for drug screening are considered among others. Scaffolds functionality depends not only on the fabrication technique, but also on the material used to build the 3D structure, the geometry and inner architecture of the structure, and the final surface properties. All being crucial parameters affecting scaffolds success. This Commentary emphasizes the importance of these parameters in scaffolds’ fabrication and also draws the attention toward the versatility of these PLA scaffolds as a potential tool in regenerative medicine and other medical fields.     

Inductive tissue engineering with protein and DNA-releasing scaffolds

Cellular differentiation, organization, proliferation and apoptosis are determined by a combination of an intrinsic genetic program, matrix/substrate interactions, and extracellular cues received from the local microenvironment. These molecular cues come in the form of soluble (e.g. cytokines) and insoluble (e.g. ECM proteins) factors, as well as signals from surrounding cells that can promote specific cellular processes leading to tissue formation or regeneration. Recent developments in the field of tissue engineering have employed biomaterials to present these cues, providing powerful tools to investigate the cellular processes involved in tissue development, or to devise therapeutic strategies based on cell replacement or tissue regeneration. These inductive scaffolds utilize natural and/or synthetic biomaterials fabricated into three-dimensional structures. This review summarizes the use of scaffolds in the dual role of structural support for cell growth and vehicle for controlled release of tissue inductive factors, or DNA encoding for these factors. The confluence of molecular and cell biology, materials science and engineering provides the tools to create controllable microenvironments that mimic natural developmental processes and direct tissue formation for experimental and therapeutic applications.     

Effects of fibroin microcarriers on inflammation and regeneration of deep skin wounds in mice

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1β, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.     

Three-dimensional printing biotechnology for the regeneration of the tooth and tooth-supporting tissues

The tooth and its supporting tissues are organized with complex three-dimensional (3D) architecture, including the dental pulp with a blood supply and nerve tissues, complex multilayer periodontium, and highly aligned periodontal ligament (PDL). Mimicking such 3D complexity and the multicellular interactions naturally existing in dental structures represents great challenges in dental regeneration. Attempts to construct the complex system of the tooth and tooth-supporting apparatus (i.e., the PDL, alveolar bone, and cementum) have made certain progress owing to 3D printing biotechnology. Recent advances have enabled the 3D printing of biocompatible materials, seed cells, and supporting components into complex 3D functional living tissue. Furthermore, 3D bioprinting is driving major innovations in regenerative medicine, giving the field of regenerative dentistry a boost. The fabrication of scaffolds via 3D printing is already being performed extensively at the laboratory bench and in clinical trials; however, printing living cells and matrix materials together to produce tissue constructs by 3D bioprinting remains limited to the regeneration of dental pulp and the tooth germ. This review summarizes the application of scaffolds for cell seeding and biofabricated tissues via 3D printing and bioprinting, respectively, in the tooth and its supporting tissues. Additionally, the key advantages and prospects of 3D bioprinting in regenerative dentistry are highlighted, providing new ideas for dental regeneration.     

Regenerative biology and medicine

The replacement of damaged tissues and organs with tissue and organ transplants or bionic implants has serious drawbacks. There is now emerging a new approach to tissue and organ replacement, regenerative biology and medicine. Regenerative biology seeks to understand the cellular and molecular differences between regenerating and non-regenerating tissues. Regenerative medicine seeks to apply this understanding to restore tissue structure and function in damaged, non-regenerating tissues. Regeneration is accomplished by three mechanisms, each of which uses or produces a different kind of regeneration-competent cell. Compensatory hyperplasia is regeneration by the proliferation of cells which maintain all or most of their differentiated functions (e.g., liver). The urodele amphibians regenerate a variety of tissues by the dedifferentiation of mature cells to produce progenitor cells capable of division. Many tissues contain reserve stem or progenitor cells that are activated by injury to restore the tissue while simultaneously renewing themselves. All regeneration-competent cells have two features in common. First, they are not terminally differentiated and can re-enter the cell cycle in response to signals in the injury environment. Second, their activation is invariably accompanied by the dissolution of the extracellular matrix (ECM) surrounding the cells, suggesting that the ECM is an important regulator of their state of differentiation. Regenerative medicine uses three approaches. First is the transplantation of cells into the damaged area. Second is the construction of bioartificial tissues by seeding cells into a biodegradable scaffold where they produce a normal matrix. Third is the use of a biomaterial scaffold or drug delivery system to stimulate regeneration in vivo from regeneration-competent cells. There is substantial evidence that non-regenerating mammalian tissues harbor regeneration-competent cells that are forced into a pathway of scar tissue formation. Regeneration can be induced if the factors leading to scar formation are inhibited and the appropriate signaling environment is supplied. An overview of regenerative mechanisms, approaches of regenerative medicine, research directions, and research issues will be given.     

Nanotechnology in regenerative medicine: the materials side

Regenerative medicine is an emerging multidisciplinary field that aims to restore, maintain or enhance tissues and hence organ functions. Regeneration of tissues can be achieved by the combination of living cells, which will provide biological functionality, and materials, which act as scaffolds to support cell proliferation. Mammalian cells behave in vivo in response to the biological signals they receive from the surrounding environment, which is structured by nanometre-scaled components. Therefore, materials used in repairing the human body have to reproduce the correct signals that guide the cells towards a desirable behaviour. Nanotechnology is not only an excellent tool to produce material structures that mimic the biological ones but also holds the promise of providing efficient delivery systems. The application of nanotechnology to regenerative medicine is a wide issue and this short review will only focus on aspects of nanotechnology relevant to biomaterials science. Specifically, the fabrication of materials, such as nanoparticles and scaffolds for tissue engineering, and the nanopatterning of surfaces aimed at eliciting specific biological responses from the host tissue will be addressed.     

Advanced biomaterials and their potential applications in the treatment of periodontal disease

Periodontal disease is considered as a widespread infectious disease and the most common cause of tooth loss in adults. Attempts for developing periodontal disease treatment strategies, including drug delivery and regeneration approaches, provide a useful experimental model for the evaluation of future periodontal therapies. Recently, emerging advanced biomaterials including hydrogels, films, micro/nanofibers and particles, hold great potential to be utilized as cell/drug carriers for local drug delivery and biomimetic scaffolds for future regeneration therapies. In this review, first, we describe the pathogenesis of periodontal disease, including plaque formation, immune response and inflammatory reactions caused by bacteria. Second, periodontal therapy and an overview of current biomaterials in periodontal regenerative medicine have been discussed. Third, the roles of state-of-the-art biomaterials, including hydrogels, films, micro/nanofibers and micro/nanoparticles, developed for periodontal disease treatment and periodontal tissue regeneration, and their fabrication methods, have been presented. Finally, biological properties, including biocompatibility, biodegradability and immunogenicity of the biomaterials, together with their current applications strategies are given. Conclusive remarks and future perspectives for such advanced biomaterials are discussed.     

Mesenchymal stem cell-based treatment for cartilage defects in osteoarthritis

Osteoarthritis (OA) is a common disorder and the restoration of the diseased articular cartilage in patients with OA is still a challenge for researchers and clinicians. Currently, a variety of experimental strategies have investigated whether mesenchymal stem cells (MSCs) instead of chondrocytes can be used for the regeneration and maintenance of articular cartilage in OA. MSCs can modulate the immune response of individuals and positively influence the microenvironment of the stem cells already present in the diseased tissue. Through direct cell–cell interaction or the secretion of various factors, MSCs can initiate endogenous regenerative activities in the OA joint. Targeted gene-modified MSC-based therapy might further enhance the cartilage regeneration in OA. Conventionally, delivery of MSCs was attained by graft of engineered constructs derived from cell-seeded scaffolds. However, intra-articular MSCs transplantation without scaffolds is a more attractive option for OA treatment. This article briefly summarizes the current knowledge about MSC-based therapy for prevention or treatment of OA, discussing the direct intra-articular injection of MSCs for the treatment of OA in animal models and in clinical applications, as well as potential future strategies for OA treatment.     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

Photocrosslinked hyaluronic acid hydrogels: natural,biodegradable tissue engineering scaffolds

Ideally, rationally designed tissue engineering scaffolds promote natural wound healing and regeneration. Therefore, we sought to synthesize a biomimetic hydrogel specifically designed to promote tissue repair and chose hyaluronic acid (HA; also called hyaluronan) as our initial material. Hyaluronic acid is a naturally occurring polymer associated with various cellular processes involved in wound healing, such as angiogenesis. Hyaluronic acid also presents unique advantages: it is easy to produce and modify, hydrophilic and nonadhesive, and naturally biodegradable. We prepared a range of glycidyl methacrylate-HA (GMHA) conjugates, which were subsequently photopolymerized to form crosslinked GMHA hydrogels. A range of hydrogel degradation rates was achieved as well as a corresponding, modest range of material properties (e.g., swelling, mesh size). Increased amounts of conjugated methacrylate groups corresponded with increased crosslink densities and decreased degradation rates and yet had an insignificant effect on human aortic endothelial cell cytocompatibility and proliferation. Rat subcutaneous implants of the GMHA hydrogels showed good biocompatibility, little inflammatory response, and similar levels of vascularization at the implant edge compared with those of fibrin positive controls. Therefore, these novel GMHA hydrogels are suitable for modification with adhesive peptide sequences (e.g., RGD) and use in a variety of wound-healing applications.     

Heparin mimetic peptide nanofibers promote angiogenesis

New blood vessel formation (angiogenesis) is one of the most important processes required for functional tissue formation. Induction of angiogenesis is usually triggered by growth factors released by cells. Glycosaminoglycans (e.g., heparan sulphates) in the extracellular matrix aid in proper functioning of these growth factors. Therefore, exogeneous heparin or growth factors were required for promoting angiogenesis in previous regenerative medicine studies. Here we report for the first time induction of angiogenesis by a synthetic nanofibrous peptide scaffold without the addition of any exogenous growth factors or heparin. We designed and synthesized a self-assembling peptide amphiphile molecule that is functionalized with biologically active groups to mimic heparin. Like heparin, this molecule has the ability to interact with growth factors and effectively enhance their bioactivity. The nanofibers formed by these molecules were shown to form a 3D network mimicking the structural proteins in the extracellular matrix. Because of heparin mimicking capabilities of the peptide nanofibers, angiogenesis was induced without the addition of exogenous growth factors in vitro. Bioactive interactions between the nanofibers and the growth factors enabled robust vascularization in vivo as well. Heparin mimetic peptide nanofibers presented here provide new opportunities for angiogenesis and tissue regeneration by avoiding the use of heparin and exogenous growth factors. The synthetic peptide nanofiber scaffolds enriched with proper chemical functional groups shown in this study can be used to induce various desired physiological responses for tissue regeneration.     

The blastema and epimorphic regeneration in mammals

Studying regeneration in animals where and when it occurs is inherently interesting and a challenging research topic within developmental biology. Historically, vertebrate regeneration has been investigated in animals that display enhanced regenerative abilities and we have learned much from studying organ regeneration in amphibians and fish. From an applied perspective, while regeneration biologists will undoubtedly continue to study poikilothermic animals (i.e., amphibians and fish), studies focused on homeotherms (i.e., mammals and birds) are also necessary to advance regeneration biology. Emerging mammalian models of epimorphic regeneration are poised to help link regenerative biology and regenerative medicine. The regenerating rodent digit tip, which parallels human fingertip regeneration, and the regeneration of large circular defects through the ear pinna in spiny mice and rabbits, provide tractable, experimental systems where complex tissue structures are regrown through blastema formation and morphogenesis. Using these models as examples, we detail similarities and differences between the mammalian blastema and its classical counterpart to arrive at a broad working definition of a vertebrate regeneration blastema. This comparison leads us to conclude that regenerative failure is not related to the availability of regeneration-competent progenitor cells, but is most likely a function of the cellular response to the microenvironment that forms following traumatic injury. Recent studies demonstrating that targeted modification of this microenvironment can restrict or enhance regenerative capabilities in mammals helps provide a roadmap for eventually pushing the limits of human regeneration.     

Recent developments in regenerative ophthalmology

Regenerative medicine(RM) is one of the most promising disciplines for advancements in modern medicine, and regenerative ophthalmology(RO) is one of the most active fields of regenerative medicine. This review aims to provide an overview of regenerative ophthalmology, including the range of tools and materials being used, and to describe its application in ophthalmologic subspecialties, with the exception of surgical implantation of artificial tissues or organs(e.g., contact lens, artificial cornea, intraocular lens, artificial retina, and bionic eyes) due to space limitations. In addition, current challenges and limitations of regenerative ophthalmology are discussed and future directions are highlighted.     

Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia

A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed.     

Preparation of adriamycin gelatin microsphere-loaded decellularized periosteum that is cytotoxic to human osteosarcoma cells

The purpose of this study was to develop a novel approach to treat bone osteosarcoma using a multipurpose scaffold aiming for local drug delivery. The slowly releasing microspheres was designed to deliver the chemotherapy drug adriamycin (ADM) and a decellularized (D) periosteum scaffold (which is known to be able to promote bone regeneration) was used to carry these microspheres. D-periosteum was obtained by physical and chemical decellularization. Histological results showed that the cellular components were effectively removed. The D-periosteum showed an excellent cytocompatibility and the ability to promote adhesion and growth of fibroblasts. Two kinds of slowly releasing microspheres, adriamycin gelatin microspheres (ADM-GMS) and adriamycin poly (dl-lactide-co-glycolide) gelatin microspheres (ADM-PLGA-GMS), were prepared and anchored to D-periosteum, resulting in two types of drug-releasing regenerative scaffolds. The effectiveness of these two scaffolds in killing human osteosarcoma cells was tested by evaluating cell viability overtime of the cancer cells cultured with the scaffolds. In summary, a gelatin/decellularized periosteum-based biologic scaffold material was designed aiming for local delivery of chemotherapy drugs for osteosarcoma, with the results showing ability of the scaffolds in sustaining release of the cancer drug and in suppressing growth of the cancer cells in vitro.     

Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.     

Monosaccharide-Responsive Phenylboronate-Polyol Cell Scaffolds for Cell Sheet and Tissue Engineering Applications

Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.     

Cementum is key to periodontal tissue regeneration: A review on apatite microstructures for creation of novel cementum-based dental implants

The cementum is the outermost layer of hard tissue covering the dentin within the root portion of the teeth. It is the only hard tissue with a specialized structure and function that forms a part of both the teeth and periodontal tissue. As such, cementum is believed to be critical for periodontal tissue regeneration. In this review, we discuss the function and histological structure of the cementum to promote crystal engineering with a biochemical approach in cementum regenerative medicine. We review the microstructure of enamel and bone while discussing the mechanism underlying apatite crystal formation to infer the morphology of cementum apatite crystals and their complex structure with collagen fibers. Finally, the limitations of the current dental implant treatments in clinical practice are explored from the perspective of periodontal tissue regeneration. We anticipate the possibility of advancing periodontal tissue regenerative medicine via cementum regeneration using a combination of material science and biochemical methods.     

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.     

Current strategies for cell delivery in cartilage and bone regeneration

Several cell-based tissue-engineering therapies are emerging to regenerate damaged tissues. These strategies use autologous cells in combination with bioresorbable delivery materials. Major functions of a delivery scaffold are to provide initial mechanical stability, homogenous three-dimensional cell distribution, improved tissue differentiation, suitable handling and properties for delivery and fixation into patients. Delivery of cells can be achieved using injectable matrices, soft scaffolds, membranes, solid load-bearing scaffolds or immunoprotective macroencapsulation. Thus, to expand the clinical potential, next generation therapies will depend on smart delivery concepts that make use of the regenerative potential of stem cells, morphogenetic growth factors and biomimetic materials.