Biological modulation and repair using plant-derived bioactives: advancements in tissue engineering and regenerative medicine

Regenerative medicine: the hurdles and hopes

In any engineered system, the understanding of the properties and interactions between the system components is of utmost importance for a successful outcome. The main components in engineered tissues are the cells, the materials used in construction of scaffolds, soluble or immobilized bioactive agents, and physical and chemical stimuli presented by the environment. As most of the mammalian tissues are constructed by bringing together repeating units of microscale complex tissue structures, understanding and control of all these components would provide the tissue engineers the capability to overcome clinical challenges as well as to develop technologies for high fidelity tissue models suitable for pharmacology, toxicology, and disease modelling applications. As tissue engineering and regenerative medicine fields mature, the level of information about how cells interact with the surrounding scaffold materials and/or other cells has increased, too. Cell microenvironment, which can be defined as the sum of all the stimuli that stem from the neighborhood of a cell and have direct or indirect effects on a given cell, has become an important consideration in exerting more control over the interactions of the cells with engineered structures. Another important aspect of tissue engineering and regenerative medicine is their temporal nature. An engineered tissue is actively remodelled over a period of time either in vitro or in vivo and currently our ability to influence this process in order to interfere with the sequence of events to achieve better regeneration is limited. However, developments in noninvasive monitoring methods and biosensor systems have begun to provide the necessary tools for tissue engineers to have real-time information about engineered tissues. This special issue set out to demonstrate the current developments and future perspectives in the use of cell microenvironment engineering and monitoring in producing functional tissues. Several investigators contributed original research or review articles about the different aspects of cell microenvironment. M.-H. Yang et al. (2014) focused on a natural biomaterial, silk fibroin, and analyzed its interaction with fibroblasts. They used proteomic approaches to understand the interaction of the cells with the substrate formed by silk fibroin in the presence of carbon nanotubes. V. Cervelli et al. (2014) demonstrated the importance of the introduction of bioactive agents in the cell microenvironment in a clinical setting in the specific example of the treatment of male pattern hair loss. They have used platelet rich plasma (PRP) injections to the scalp and observed a significant increase in hair density, epithelial thickness, and number of hair follicles. W.-Y. Lin et al. (2014) studied another important aspect of cell microenvironment, namely, the effect of dynamic mechanical stress/strain conditions. They have developed a system which can apply cyclic compressive stress to chondrocytes at physiologically relevant levels. They have shown that the application of stress has a direct effect on the chondrocyte metabolic activity and glycosaminoglycan secretion. Another aspect of cartilage tissue engineering, the extracellular matrix/chondrocyte interactions, was reviewed by Gao et al. (2014), where they focused on the signaling pathways that are active during chondrogenesis. In another review paper, Barthes et al. (2014) gave a comprehensive description of cell microenvironment and how each component can be used to direct cellular activity for tissue engineering applications, together with the current developments in the monitoring of artificial tissues. The advances in tissue engineering not only established it as a field where solutions to serious clinical problems can be developed but also as a growing area where enabling technologies such as organ-on-a-chip systems for pharmacological purposes can be devised. In order to have models accurately mimicking artificial organs, it is important to have a good grasp of the specific microenvironments pertaining to each tissue type. We believe engineering of the cell microenvironment will be an important part of future tissue engineering activities. Nihal Engin Vrana Vasif Hasirci Garrett Brian McGuinness Albana Ndreu-Halili

Peripheral nerve injuries account for 4 % of all injuries, and the consequences of trauma are a major medical and social problem, since they are characterized by a significant and long-term decline in limb function, and a high level of disability in patients. According to our data, up to 40% of patients sought specialized care for more than 6 months after the injury, and 19.9% were treated conservatively for an unreasonably long period of time. It led to an increase in the portion of unsatisfactory treatment results, since the prognosis of the further functional and useful degree of nerve recovery worsens with increasing time after injury. The main objective was to select the optimal option of biocompatible material for implementation in practice in case of traumatic peripheral nerve damage. Materials and methods. The analysis of medical literature for 2015–2020 was conducted. First of all, it should be noted that modern non-biological resorbable tubes are made of polyglycolic and polylactic acids. Non-resorbable tubes, including silicone, have shown undesirable effects, including axon compression during regeneration and the reaction of a fibrous foreign body. Hollow cylindrical tubes can be manufactured in several ways, such as electrospinning, crosslinking, physical film rolling, injection molding, melt extrusion, and braiding. Adequate surgical treatment of peripheral nerve injuries requires that the surgeon, in addition to an accurate knowledge of the anatomical details of the affected area, would also be familiar with microsurgical methods and had necessary equipment to operate. The main procedure in peripheral nerve surgery is the restoration of nerve continuity, which can be obtained by direct coaptation between the two ends of a severed nerve or by the introduction of nerve grafts to replace a defect in nerve tissue. Polyester is the most common synthetic material used in neural tissue engineering, along with polylactic acid, polycaprolactone, and polyglycolic acid. In combination with mesenchymal stem cells of the bone marrow, polylactic acid showed better results and accelerated the recovery of peripheral nerves. Polylactic acid directed the migration of Schwann's cells and induced the formation of a normal nervous structure. It was proved that the polycaprolactone material had an effect similar to that of autografts in nerve repair, and its characteristics were better than in a polylactic acid tube. Polyglycolic acid also possesses sufficient mechanical properties and can be used to repair a nerve defect. Artificial synthetic materials have good biocompatibility and biodegradability with minimal toxicity. For the production of high-purity polymer monomers, which are necessary for the manufacture of the frame, much time and financial costs are required. Moreover, the elasticity and hardness of such materials are imperfect. Three main natural biomaterials are used in tissue repair: collagen, silk, and gelatin. Collagen tube is the most widely used biological material in clinical practice. Silk materials with the protein fibroin, which promote the release of certain substrates, such as nerve growth factor particles, and provide more nutrients and a more favorable microenvironment for nerve repair, are worth noticing. Silk fibroin has good compatibility with the neurons of the dorsal root ganglia and supports cell growth. Gelatin materials are preferred due to the reduction of micromanipulation during nerve recovery. Natural biomaterials are easy to obtain in sufficient quantities; they have good biocompatibility and biodegradability and are easily absorbed by the body. However, each natural biomaterial has its drawbacks. Some of them are brittle or break down in a humid environment. Some natural materials are insoluble in water and traditional organic solvents, which limits their use. One of the most widely used biopolymers of natural origin is chitosan. Chitosan, derived by chitin deacetylation, plays a supporting, protective, and guiding role in the early stage of recovery of peripheral nerves and can provide a relatively stable, localized microenvironment during regeneration. Chitosan is absorbed and gradually decomposed in the late phase of recovery and regeneration of the nervous system. Issues regarding graphene-based nanomaterials use are considered. Graphene is a two-dimensional carbon nanomaterial with good optical, electrical and mechanical properties. It should be noted that when graphene nanoparticles incorporate into a chitosan or gelatin frame and are used to repair peripheral nerve damage in rats, this has contributed to the regeneration of the damaged nerve more quickly. Graphene also reduced the inflammatory response and accelerated the migration of endogenous neuroblasts. Hence, the use of these materials is not well understood due to the significant duration of recovery of the denervated proximal end of the nerve, so further research is needed to identify the advantages or disadvantages of their use.

Regenerative medicine has received potential attention around the globe, with improving cell performances, one of the necessary ideas for the advancements of regenerative medicine. It is crucial to enhance cell performances in the physiological system for drug release studies because the variation in cell environments between in vitro and in vivo develops a loop in drug estimation. On the other hand, tissue engineering is a potential path to integrate cells with scaffold biomaterials and produce growth factors to regenerate organs. Scaffold biomaterials are a prototype for tissue production and perform vital functions in tissue engineering. Silk fibroin is a natural fibrous polymer with significant usage in regenerative medicine because of the growing interest in leftovers for silk biomaterials in tissue engineering. Among various natural biopolymer-based biomaterials, silk fibroin-based biomaterials have attracted significant attention due to their outstanding mechanical properties, biocompatibility, hemocompatibility, and biodegradability for regenerative medicine and scaffold applications. This review article focused on highlighting the recent advancements of 3D printing in silk fibroin scaffold technologies for regenerative medicine and tissue engineering.

Regenerating injured nerves is difficult because they have little spontaneous regeneration potential. Advances in tissue engineering and regenerative medicine have emphasized the possibility of biomaterial-based methods for nerve healing. Natural protein-based biomaterials have benefits over synthetic ones, such as biocompatibility, non-immunogenicity, and biodegradability. Silk fibroin, generated from mulberry and non-mulberry silkworms, is especially promising because of its abundance, simplicity of processing into nerve-like structures, adjustable biodegradability, and mechanical robustness. Furthermore, non-mulberry silk fibroin contains the cell-affinitive RGD tripeptide, which enhances its ability to repair nerves. Studies using silk fibroin (SF)--based nerve conduits have demonstrated nerve regeneration rates of up to 80–90% compared to autografts, which remain the clinical gold standard. SF conduits exhibit outstanding mechanical properties, with tensile strengths up to 300 MPa and elastic moduli adjustable between kPa-MPa range, which closely mimic the native tissue and ensure durability in dynamic environments. This review explores the diverse types of silkworm silk fibroin (SSF) and their applications in biomaterial-based Peripheral Nerve Repair (PNR). It discusses the integration of SSF with other biopolymers and synthetic polymers, highlighting advancements in nerve guidance channels incorporating electro-conductive materials to enhance regeneration rates. The literature search was primarily conducted using the Web of Science database, employing relevant keyword combinations such as “silk fibroin + nerve repair,” “silk fibroin + peripheral nerve repair,” “silk + nerve repair,” and “silk + nerve repair + electrical stimulation.” As this review focuses on silkworm silk-based biomaterials, studies involving spider silk or recombinant silk-based biomaterials were excluded. The period considered began with the earliest relevant studies, with an emphasis on more recent advancements up to November 2024 to capture the latest developments in the field. Identified studies were categorized based on the biomaterial composition, including pure silk biomaterials, silk biopolymer binary composites, silk synthetic binary composites, and silk-hybrid composites. Key findings were synthesized to highlight the progress, challenges, and future directions in applying silk fibroin-based scaffolds and electrical stimulation technologies for nerve repair. The findings provide insights into the potential of SSF-based biomaterials and propose future directions for developing advanced nerve repair strategies.

Background Regenerative Medicine (RM) is a branch of medicine that aims to regenerate tissues and organs to overcome the problems transplants entail (poor availability, risk of rejection and intense immunosuppression). To do this, RM makes use of tissue engineering (TE). This fundamental branch deals with creating biological scaffolds capable of performing the role that physiologically belongs to the extracellular matrix (ECM). In this review, we report how specific characteristics of the scaffolds (bio-compatibility, biodegradability and mechanical and conformal properties) can be obtained using 3D printing, which facilitates the emulation of physiological tissues and organs. Purpose and scope This review reports recent advances in the fabrication method of bioactive scaffolds that can be used clinically, providing support for cell seeding and proliferation. To this end, silk fibroin, tannin and graphene were used to improve the scaffold’s electro-bio-mechanical properties. These materials in different compositions are studied to demonstrate their potential use as bio-ink in bioadhesives and cellularized and implantable 3D-printed scaffolds. Summary of new synthesis and conclusions reached in the review Silk fibroin is a natural biopolymer; tannin, on the other hand, is a biological polyphenol, highly reactive with other molecules by nature and with promising antioxidant capabilities. Finally, graphene is nothing more than a monolayer of graphite that has been shown to implement the mechanics and electrical conductivity of the compounds in which it is inserted; it also has excellent biocompatibility and surface area, qualities that promote cell adhesion and growth. Conclusion Polyphenols and graphene have been shown to work in synergy in improving the electro-mechanical properties of silk fibroin scaffolds. We reported optimal and potentially market-competitive bioadhesives, but above all, the proliferation of neuronal precursor cells in vitro was successfully demonstrated.

The International Conference on Bioprinting and Biofabrication in Bordeaux (3B'09) demonstrated that the field of bioprinting and biofabrication continues to evolve. The increasing number and broadening geography of participants, the emergence of new exciting bioprinting technologies, and the attraction of young investigators indicates the strong growth potential of this emerging field. Bioprinting can be defined as the use of computer-aided transfer processes for patterning and assembling living and non-living materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacokinetic and basic cell biology studies. The use of bioprinting technology for biofabrication of in vitro assay has been shown to be a realistic short-term application. At the same time, the principal feasibility of bioprinting vascularized human organs as well as in vivo bioprinting has been demonstrated. The bioprinting of complex 3D human tissues and constructs in vitro and especially in vivo are exciting, but long-term, applications. It was decided that the 5th International Conference on Bioprinting and Biofabrication would be held in Philadelphia, USA in October 2010. The specially appointed 'Eploratory Committee' will consider the possibility of turning the growing bioprinting community into a more organized entity by creating a new bioprinting and biofabrication society. The new journal Biofabrication was also presented at 3B'09. This is an important milestone per se which provides additional objective evidence that the bioprinting and biofabrication field is consolidating and maturing. Thus, it is safe to state that bioprinting technology is coming of age.

Silk Fibroin Microfluidic Devices.

Regenerative medicinal products were dealt with fewer than two categories only, pharmaceutical products and medical devices in the previous Pharmaceutical Affairs Law (PAL). Because regenerative medicinal products could not fully fit within the conventional regulatory contexts for pharmaceuticals, they were less available to the public. To overcome this issue, the Japanese parliament has legislated Laws for the Promotion of Comprehensive Measures to Facilitate Swift Distribution and Safe Use of Regenerative Medicines available to the Public (Regenerative Medicine Promotion Act). The Japanese government has changed the regulatory framework for regenerative medicinal products, 1) upgrading of directives for human stem cell-based clinical research to the Law concerning the Establishment of Safety of Regenerative Medicine Materials (Regenerative Medicine Safety Assurance Act), and 2) setting a new category for regenerative medicinal products in the Pharmaceutical and Medical Device Act (PMD Act), the latest revised PAL. It is noteworthy that the PMD Act permits expedited, conditional, and time-limited marketing approval as an exception for non-homogeneous regenerative medicinal products when efficacy is anticipated while safety is demonstrated. With these revisions of the regulatory framework for regenerative medicinal products, two tracks have become available for societal contribution to regenerative medicines. Japan is the only country where society has these two tracks, and social experiments on regenerative medicines in spectacular proportion are launched for societal contribution. We review here the history of the struggle to accelerate the availability of regenerative medicinal products for patients in Japan.

Editorial: Tissue Engineering: Perspectives, Challenges, and Future Directions

History of Tissue Engineering and A Glimpse Into Its Future

Silk fibroin for skin injury repair: Where do things stand?

To the Editor: The article by Xue et al1 on human embryonic stem cell-derived pacemakers illustrates that embryonic stem cells differentiated into spontaneously beating cardiocytes may function as biological pacemakers and mentions a potential limitation: The intrinsic pacemaker rate was slower than desirable. They suggest that incorporating HCN pacemaker channel genes might achieve more desirable rates, an idea consistent with our published results using HCN2 in gene- and adult human mesenchymal stem cell (hMSC)-based therapies.2–4 However, certain of the comments by Xue et al misinterpret our own work on HCN-loaded hMSCs. They state that “…these modified, undifferentiated, human mesenchymal stem cells are incapable of pacing quiescent cells because the former are neither electrically active nor genuine cardiac cells” (p 19). This statement suggests a misunderstanding of the rationale and underlying biophysics of the hMSC experiments. In fact, generation of pacemaker activity does not require delivery of an “excitable differentiated cardiac cell,” but only that the delivered cell (1) carry sufficient pacemaker current and (2) make gap junctions; thus, the hMSC-myocyte pair should behave as a pacemaker unit entirely equivalent to a single heart cell with substantial if. We clearly demonstrated both …

This study aims to produce composite nanofibers from polylactic acid (PLA) and silk fibroin (SF) with mechanical properties similar to that of a native meniscus. SF is a natural protein, well known for its good biocompatibility and biodegradability but it has poor mechanical properties compared to synthetic polymers. SF contains proteins which can promote cell adhesion and cell proliferation required for a tissue engineering scaffold. PLA is a popular material that is widely used in tissue engineering. It provides high mechanical properties but lacks bioactivity and cell affinity. Thus, a PLA/SF composite scaffold can improve these drawbacks. In this study, PLA/SF nanofibers were fabricated using an electrospinning process at various PLA and SF ratios (PLA: SF 100:0, 75:25, 50:50, and 25:75). The SF content in the emulsion dominated the fiber diameter, fiber arrangement and processibility of nanofibrous scaffold. With increasing SF content, the %elongation at break of PLA/SF scaffold increased but Young’s modulus decreased. The wettability of electrospun PLA was increased with an increase in SF content.

A mere glance at the foundation of the sericulture industry to produce silk and the consequent establishment of the Silk Road to transport it; elucidates the significant role that this material has played in human history. Owing to its exceptional robustness, silk was introduced into medicine as a surgical suture approximately two millennia ago. During the last decades, silk has garnered attention as a possible source of biological-based materials that can be effectively used in regenerative medicine. Silk's unique characteristics, like its low immunogenicity, suitable adhesive properties, exceptional tensile strength, perfect hemostatic properties, adequate permeability to oxygen and water, resistance to microbial colonization, and most importantly, excellent biodegradability; make it an outstanding choice for biomedical applications. Although there are many different types of silk in nature,Bombyx mori(B. mori) silk accounts for about 90% of global production and is the most thoroughly investigated and the most commonly used. Silk fibroin (SF) and silk sericin (SS) are the two main protein constituents of silk. SF has been manufactured in various morphologic forms (e.g. hydrogels, sponges, films, etc) and has been widely used in the biomedical field, especially as a scaffold in tissue engineering. Similarly, SS has demonstrated a vast potential as a suitable biomaterial in tissue engineering and regenerative medicine. Initial studies on SF and SS as wound dressings have shown encouraging results. This review aims to comprehensively discuss the potential role of silk proteins in refining wound healing and skin regeneration.