Materials For Regenerative Medicine Research Articles

Long-term culture of rat hepatocytes using human amniotic membrane as a culture substrate.

Amniotic membrane is attracting attention as a new material for regenerative medicine. We herein report that the culture of primary rat hepatocytes on human amniotic membrane maintained their morphology and their production of albumin for at least two months. Human amniotic membrane was collected during planned cesarean section and kept frozen until usage. Primary rat hepatocytes were plated on human amniotic membrane. Hepatocytes accumulated as colonies on amniotic membrane, and their rat albumin level was maintained for two months. Their three-dimensional structure on extracellular matrix, which is abundant in amniotic membranes might influence the maintenance of the hepatocyte-specific function.

Manufacturing of Human Tissues as off-the-Shelf Grafts Programmed to Induce Regeneration.

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes.

The balanced microenvironment regulated by the degradants of appropriate PLGA scaffolds and chitosan conduit promotes peripheral nerve regeneration.

Tissue-engineered nerve grafts (TENGs) are the most promising way for repairing long-distance peripheral nerve defects. Chitosan and poly (lactic-co-glycolic acid) (PLGA) scaffolds are considered as the promising materials in the pharmaceutical and biomedical fields especially in the field of tissue engineering. To further clarify the effects of a chitosan conduit inserted with various quantity of poly (lactic-co-glycolic acid) (PLGA) scaffolds, and their degrades on the peripheral nerve regeneration, the chitosan nerve conduit inserted with different amounts of PLGA scaffolds were used to repair rat sciatic nerve defects. The peripheral nerve regeneration at the different time points was dynamically and comprehensively evaluated. Moreover, the influence of different amounts of PLGA scaffolds on the regeneration microenvironment including inflammatory response and cell state were also revealed. The modest abundance of PLGA is more instrumental to the success of nerve regeneration, which is demonstrated in terms of the structure of the regenerated nerve, reinnervation of the target muscle, nerve impulse conduction, and overall function. The PLGA scaffolds aid the migration and maturation of Schwann cells. Furthermore, the PLGA and chitosan degradation products in a correct ratio neutralize, reducing the inflammatory response and enhancing the regeneration microenvironment. The balanced microenvironment regulated by the degradants of appropriate PLGA scaffolds and chitosan conduit promotes peripheral nerve regeneration. The findings represent a further step towards programming TENGs construction, applying polyester materials in regenerative medicine, and understanding the neural regeneration microenvironment.

Involvement of somatic stem cells in encapsulation of foreign–body reaction in canine subcutaneous Biotube tissue formation

Generally, the thickness of tubular tissues formed from silicone rods through encapsulation of the foreign-body reaction is less than approximately 0.2mm. On the other hand, it is unclear how hollow cylindrical molds can provide thick tubular tissues, known as Biotubes, with a thickness exceeding 1mm, during in-body tissue architecture (iBTA) using encapsulation. In this study, histological and structural analyses were performed to understand the reason for the formation of thick mold-based Biotubes. Molds were assembled with a gap between a silicone rod and a stainless-steel cylinder and were embedded into the dorsal subcutaneous pouches of beagles for 2 or 4 weeks. Thick Biotubes were obtained from the harvested mold. The histological analysis showed that the lumen side of the thick Biotubes consisted primarily of type I collagen fibers and α-smooth muscle actin-positive cells, similar to the original rod-based thin Biotubes formed only from silicone rods. Interestingly, the outer region of the thick Biotubes was an immature connective tissue consisting of type III collagen, including primitive somatic stem cells expressing CD90 and SSEA4. These stem cells may have contributed to the formation of the thick-walled Biotubes by differentiating into other cell types and through growth factor production. Because of the potential tissue-repair ability of these stem cells, iBTA could be useful for elucidating the regeneration process, remodeling the physiology/pathology of tissue defects/damage, and cell acquisition. This technology can provide autologous stem cells without invitro cell culture. Moreover, thick-walled Biotubes may be useful as an alternative stem cell-containing material in regenerative medicine.

Decellularized natural 3D cellulose scaffold derived from Borassus flabellifer (Linn.) as extracellular matrix for tissue engineering applications

In this study, Borassus flabellifer (Linn.) (BF) immature endosperm was decellularized to produce three dimensional (3D) cellulose scaffolds that can support mammalian 3D cell culture. To this regard, we first evaluated the chemical composition, nutritive profile and pharmacological activities of BF endosperm. The results demonstrated that the BF tissue represented a complex concoction of polysaccharides with intrinsic phyto-ingredients which provide excellent pharmacological properties. Furthermore cellulosic scaffolds (CS) obtained from BF was treated with chitosan to produce cellulose-chitosan (CS/CHI) hybrid scaffolds. The comparative investigation on both scaffolds exhibited adequate swelling with controlled porosity and pore-size distribution. The physiochemical characterization showed reduced biodegradation, improved thermal stability and enhanced compressive strength in CS/CHI group. Biological studies reported favorable adhesion and proliferation of fibroblasts with evident cellular penetration and colonization on the both scaffolds. Taken together, plant derived cellulosic scaffolds could be used as an alternative scaffolding material in regenerative medicine.

Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation‐inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin‐like growth factor with a key amino acid (3,4‐dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid‐phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long‐lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Insights into the Role of Biopolymer Aerogel Scaffolds in Tissue Engineering and Regenerative Medicine.

The global transplantation market size was valued at USD 8.4 billion in 2020 and is expected to grow at a compound annual growth rate of 11.5% over the forecast period. The increasing demand for tissue transplantation has inspired researchers to find alternative approaches for making artificial tissues and organs function. The unique physicochemical and biological properties of biopolymers and the attractive structural characteristics of aerogels such as extremely high porosity, ultra low-density, and high surface area make combining these materials of great interest in tissue scaffolding and regenerative medicine applications. Numerous biopolymer aerogel scaffolds have been used to regenerate skin, cartilage, bone, and even heart valves and blood vessels by growing desired cells together with the growth factor in tissue engineering scaffolds. This review focuses on the principle of tissue engineering and regenerative medicine and the role of biopolymer aerogel scaffolds in this field, going through the properties and the desirable characteristics of biopolymers and biopolymer tissue scaffolds in tissue engineering applications. The recent advances of using biopolymer aerogel scaffolds in the regeneration of skin, cartilage, bone, and heart valves are also discussed in the present review. Finally, we highlight the main challenges of biopolymer-based scaffolds and the prospects of using these materials in regenerative medicine.

Silk Fibroin: An Ancient Material for Repairing the Injured Nervous System.

Silk refers to a family of natural fibers spun by several species of invertebrates such as spiders and silkworms. In particular, silkworm silk, the silk spun by Bombyx mori larvae, has been primarily used in the textile industry and in clinical settings as a main component of sutures for tissue repairing and wound ligation. The biocompatibility, remarkable mechanical performance, controllable degradation, and the possibility of producing silk-based materials in several formats, have laid the basic principles that have triggered and extended the use of this material in regenerative medicine. The field of neural soft tissue engineering is not an exception, as it has taken advantage of the properties of silk to promote neuronal growth and nerve guidance. In addition, silk has notable intrinsic properties and the by-products derived from its degradation show anti-inflammatory and antioxidant properties. Finally, this material can be employed for the controlled release of factors and drugs, as well as for the encapsulation and implantation of exogenous stem and progenitor cells with therapeutic capacity. In this article, we review the state of the art on manufacturing methodologies and properties of fiber-based and non-fiber-based formats, as well as the application of silk-based biomaterials to neuroprotect and regenerate the damaged nervous system. We review previous studies that strategically have used silk to enhance therapeutics dealing with highly prevalent central and peripheral disorders such as stroke, Alzheimer’s disease, Parkinson’s disease, and peripheral trauma. Finally, we discuss previous research focused on the modification of this biomaterial, through biofunctionalization techniques and/or the creation of novel composite formulations, that aim to transform silk, beyond its natural performance, into more efficient silk-based-polymers towards the clinical arena of neuroprotection and regeneration in nervous system diseases.

Stromal-vascular fraction of adipose tissue as an alternative source of cellular material for regenerative medicine

Adipose tissue is the most convenient source of cellular material for regenerative medicine as it can be obtained in significant quantities via cosmetic liposuction, lipoaspiration of subcutaneous fat or by excision of fat deposits. Adipose tissue consists of adipocytes and cells, which are the part of the stromal-vascular fraction (SVF). Different cell populations can be isolated from SVF, among which the population of adipose tissue stem cells (adipose-derived stem cells, ADSC) is especially important for regenerative medicine. SVF can be obtained relatively easily from adipose tissue (adipose tissue is an alternative to bone marrow in terms of being a source of stem cells) and used to treat various pathologies. Recent studies show that SVF not only has a therapeutic effect similar to that of ADSC, but in some cases is even more effective. The article provides the analysis of the main methods of SVF obtainment, characteristics of SVF cellular composition, its potential for use in clinical medicine and its main advantages over other sources of cellular material, including­ ADSC cultured in vitro, for regenerative medicine. Keywords: adipocytes, adipose-derived stem cells, regenerative medicine, stromal-vascular fraction

Bioactive Polymeric Materials for the Advancement of Regenerative Medicine.

Biopolymers are widely accepted natural materials in regenerative medicine, and further development of their bioactivities and discoveries on their composition/function relationships could greatly advance the field. However, a concise insight on commonly investigated biopolymers, their current applications and outlook of their modifications for multibioactivity are scarce. This review bridges this gap for professionals and especially freshmen in the field who are also interested in modification methods not yet in commercial use. A series of polymeric materials in research and development uses are presented as well as challenges that limit their efficacy in tissue regeneration are discussed. Finally, their roles in the regeneration of select tissues including the skin, bone, cartilage, and tendon are highlighted along with modifiable biopolymer moieties for different bioactivities.

Polarization of Stem Cells Directed by Magnetic Field-Manipulated Supramolecular Polymeric Nanofibers.

Precise assembly of the cytoskeleton (e.g., actin, tubulin, and intermediate filaments) is of great importance for stem cell polarization and tissue regeneration. Recently, artificial manipulation of cytoskeleton assembly for remodeling stem cell polarization and ultimate cell fates attracts more and more interest of both chemists and biologists. Herein, we report the magnetic field-directed formation of biocompatible supramolecular polymeric nanofibers composed of two subunits: a β-cyclodextrin-bearing hyaluronic acid host polymer (HACD) and magnetic nanoparticles modified with actin-binding peptide and adamantane (MS-ABPAda). Transmission electron microscopy indicated that when HACD and MS-ABPAda were exposed to a magnetic field, they self-assembled into long nanofibers along the direction of the magnetic field, and the rate of nanofiber formation was linearly correlated with the strength of the magnetic field. Interestingly, when incubated with dental pulp stem cells, the nanofibers specifically drove tip extension and polarization of the cells, a phenomenon that can be attributed to targeting of actin-binding peptide to the actin cytoskeleton and subsequent polarization of the nanofibers. The successful application of these magnetic field-responsive supramolecular polymers on accurately driving polarization of mammalian cells is expected to be of great value for artificially manipulating cell fate and developing intelligent responsive materials in regenerative medicine.

Novel applications of platelet concentrates in tissue regeneration (Review).

Numerous studies have explored the suitability of biocompatible materials in regenerative medicine. Platelet concentrates are derived from centrifuged blood and are named according to their biological characteristics, such as platelet-rich plasma, platelet-rich fibrin and concentrated growth factor. Platelet concentrates have gained considerable attention in soft and hard tissue engineering. Indeed, multiple components of autologous platelet concentrates, such as growth factors, fibrin matrix and platelets, serve essential roles in wound healing. Current studies are focused on cutting-edge strategies to meet the requirements for tissue restoration by improving the properties of autologous platelet concentrates. In the present review, applications of platelet concentrates for tissue engineering are discussed, presenting a selection of recent advances and novel protocols. In addition, several aspects of these strategies, such as the advantages of lyophilized platelet concentrates and the combination of platelet concentrates with biomaterials, stem cells or drugs are discussed. The present review aims to summarize novel strategies using platelet concentrates to improve the outcomes of wound healing.

Synchrotron Fourier transform infrared microspectroscopy (sFTIRM) analysis of unfolding behavior of electrospun collagen nanofibers

Collagen nanofibers are popular extracellular matrix (ECM) materials in regenerative medicine. Electrospinning of collagen dissolved in organic solvents is widely used for fabricating anisotropic collagen nanofibers; however, such fibers are water-soluble and require cross-linking before use as scaffolds for cell culture. Herein, in-situ crosslinking during electrospinning process is suggested by using different chemical agents, namely genipin and glutaraldehyde, and physical crosslinking method (UV light). sFTIRM; Synchrotron Fourier-Transform Infrared Microspectroscopy is a powerful tool that sheds light on the molecular structure of collagen nanofibers. Applied extraction methods caused shifts on protein band positions. Electrospinning process prevents self-assembly of collagen molecules and obtained electrospun collagen nanofibers have lower band positions. Crosslinkers have effect on the secondary structure of collagen molecules. Among different crosslinkers, genipin in-situ crosslinking process perform better in preserving the native structure of electrospun collagen nanofibers than the physical crosslinking method (UV).

Living Materials for Regenerative Medicine

Abstract Regenerative medicine has been attracting tremendous attention during the past few decades because it is promising in overcoming the limitations of donor shortage and immune complications in direct transplantations. The ongoing progress in this field calls for the rapid growth of living materials, which consist of live biological agents and can be designed together with synthetic materials to meet the application demands of regenerative medicine. In this review, we present a summary of the state-of-the-art progress of living materials that are applied in regenerative medicine. We first introduce the advanced engineering approaches that are employed to prepare living materials containing live cells, typically including genetic engineering, cell coating, microfluidics, and bioprinting, etc. Afterwards, we enumerate different application aspects of living materials in regenerative medicine, including tissue scaffold, cell therapy, tissue models, and so on. Finally, we give a concise conclusion and provide a perspective of this field.

ІННОВАЦІЇ В НАНОМЕДИЦИНІ ТА НАНОФАРМАЦІЇ

The review examines current trends in the development of biomedical nanotechnology, its application in various fields of medicine and prospects for further development. Among the main directions — monitoring and control of delivery, distribution and use of drugs; nanobiosensors; diagnostics of pathological states; theranostics; use of nanostructured materials in regenerative medicine; nanotoxicology; nanophytopharmacy; green synthesis; development of new nanobiotech devices and nanorobototics.

Functional and cell surface characteristics of periodontal ligament cells (PDLCs) on RGD-synthetic polypeptide conjugate coatings.

Periodontal ligament cells (PDLCs) are an important source for periodontal tissue healing and regeneration. Proper cell adhesion is a key for survival of anchorage-dependent cells and also initiates further intracellular signals for essential cellular functions. We aimed to test 3 different synthetic conjugates with integrin-binding RGD sequence (SAK-c[RGDfC], AK-c[RGDfC], and SAK-opn on the adhesion of human PDLCs and subsequent events including proliferation, migration, behavior of cell surface molecules, and osteogenic differentiation. Synthetic peptides were synthesized by solid-phase technique and attached to branched chain polymeric polypeptides via thioether linkage. Simple adsorption method was used to coat tissue culture plastic or electric arrays. PDLCs were isolated from 24 surgically extracted human third molars. Cell adhesion and proliferation were measured with real-time impedimetric xCELLigence SP system. Cell migration assay was performed with Ibidi® Culture inserts. Cell surface antigens were detected using flow cytometry analysis. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red S staining, and real-time qPCR was performed to analyze the osteoblast-related gene expression. Osteogenic differentiation and adipogenic differentiation of PDLCs were monitored by real-time Electrical Cell-Substrate Impedance Spectroscopy (ECIS). Primary outcome of this study relies on that all three synthetic RGD peptides improved PDLC adhesion (P<.05). When animal serum is absent in culture medium, SAK-c[RGDfC] and AK-c[RGDfC] elevated cell adhesion (P<.05). Cell migration was enhanced by SAK-c[RGDfC] and AK-c[RGDfC] (P<.05). After 1-week treatment, all synthetic peptides elevated CD105 (1.7- to 2.2-fold) and CD146 (1.3- to 1.5-fold) markers and caused different integrin patterns. ALP activity (1.4-fold) and ARS (1.8- and 2.0-fold) were increased by SAK-c[RGDfC] and AK-c[RGDfC] in absence of osteogenic supplements, and all the peptides supported the mineralization under osteogenic condition (P<.05). RT-qPCR revealed the upregulation of bone sialoprotein (5.0- to 7.8-fold), osteocalcin (2.3- to 2.7-fold), and ALP (1.9- to 2.3-fold) gene expression in osteogenesis-induced PDLCs. ECIS monitoring showed that higher impedance was generated by the osteogenic induction compared with the adipogenic or the non-induced (P<.05). Our study demonstrates that SAK-c[RGDfC] and AK-c[RGDfC] improved adhesion and migration of PDLCs and supported osteogenic differentiation of PDLCs. These cyclic RGD peptides proved to be applicable biocompatible material in regenerative medicine.

Development of chitinous nanofiber-based flexible composite hydrogels capable of cell adhesion and detachment

Chitinous nanofibers (NFs) exhibit a specific physiological activity not observed in the powdered form. While three-dimensional cell culture scaffolds are being actively studied, attempts have been made to use chitinous materials as well. In this study, we aimed to develop a cell-adhesive chitinous NF-based flexible hydrogel. Such a gel will be easy to handle, and there is a possibility that the gel will be able to be three-dimensionally molded. For this purpose, the surface of chitinous NFs was modified to introduce vinyl polymerizable functional groups, and the modified NFs were copolymerized with an N-isopropylacrylamide (NIPAM) monomer in an aqueous system. First, by modification of chitosan NFs with maleic anhydride (MA), a flexible gel was obtained by subsequent copolymerization with NIPAM. However, the adhesion of L929 mouse fibroblasts onto MA-modified NFs was extremely lower than that of chitosan NFs, probably due to the simultaneous introduction of anionic groups. The desired cell-adhesive flexible hydrogel was eventually obtained by introducing cationic groups and polymerizable functional groups on the chitinous NF surface. Cells adhered to the gel could be detached by cooling to 4 °C. The composite hydrogel fabricated here may be useful as a cell culture scaffold material in regenerative medicine. Chitinous nanofibers (NFs) exhibit a specific physiological activity not observed in the powdered form. Herein, we aimed to develop a cell-adhesive chitinous NF-based flexible hydrogel. The desired cell-adhesive flexible hydrogel was obtained by introducing cationic groups and vinyl polymerizable functional groups on the surface deacetylated chitin NFs as well as copolymerizing it with N-isopropylacrylamide monomer in an aqueous system. Cells adhered to the gel could be detached by cooling to 4 °C. The composite hydrogel fabricated here may be useful as a cell culture scaffold material in regenerative medicine.

Study of polylactide matrices using x-ray microtomography

Three-dimensional matrices of biodegradable polymers are promising materials for regenerative medicine. They are widely used in restoring the integrity and functions of tissues and organs using bio-artificial tissue engineering structures. We present the results of studying the structure of porous bioresorbable polymer matrices for tissue engineering using X-ray microtomography. Samples were obtained by supercritical fluid plasticization of D,L-polylactide with subsequent foaming in cylindrical molds. The tomographic method makes it possible to construct a three-dimensional voxel model of the object under study and gain apart from the estimate on the integral matrix porosity (characteristic data obtained by traditional sorption procedures) additional information about the size and spatial distribution of pores thus providing a possibility of optimization of the process parameters for production of polylactide matrices required for specific biomedical applications of architectonics, as well as forecasting the processes of their bioresorption in enzymatic media. The experiments were carried out on a laboratory microtomograph (Mo anode, the scan time of the sample is 120 min, the detector pixel size is 9 μm). Tomographic reconstruction was performed by algebraic method. The binarization procedure required for calculation of the structural characteristics of studied matrices was implemented by the method with the choice of a global threshold. Calculations of the porosity and homogeneity of the porosity distribution in the bulk, as well as estimation of the specific surface area of pores revealed the isotropy of the spatial structure of polylactide matrices.

Shape-dependent regulation of differentiation lineages of bone marrow-derived cells under cyclic stretch

Multipotent stem cells are considered as a key material in regenerative medicine, and the understanding of the heterogeneity in the differentiation potentials of bone marrow-derived cells is important in the successful regenerative tissue repair. Therefore, the present study has been performed to investigate how the differentiation of post-harvest, native bone marrow-derived cells is regulated by cyclic stretch in vitro. Bone marrow-derived cells were obtained from mouse femur of both hind limbs and categorized into the following five categories: amebocytes, round cells, spindle cells, stellate cells and others. The cells were seeded on a silicone-made stretch chamber, and subjected to cyclic stretch with an amplitude of 10% at a frequency of 1 Hz for 7 days for cell shape analysis and for 3 days for the analysis of the expression of marker proteins of osteogenic (osteocalcin), vascular smooth muscle (α-smooth muscle actin and smooth muscle myosin heavy chain) and neurogenic (neurofilament) differentiation. When disregarding the differences in the cell shapes, there was an overall trend that the application of 10% cyclic stretch inhibited osteogenic and neurogenic differentiation, but enhanced smooth muscle differentiation. Close examinations revealed that round cells were influenced the most by cyclic stretch (significant up- or down-regulation in all the four marker protein expressions) while amebocytes and spindle cells were only influenced by cyclic stretch for vascular smooth muscle and/or neurogenic differentiation. As far as the authors know, this is the first study reporting the shape-related differences in the fate decision criteria for mechanical strain in bone marrow-derived cells.

Transcriptomic profiling of neural stem cell differentiation on graphene substrates

Graphene exhibits excellent mechanical strength, electrical conductivity and good biocompatibility, which make it a suitable candidate as a neural interfacing material in regenerative medicine and tissue engineering. Graphene is reported to promote both of neural stem cells (NSCs) proliferation and differentiation. However, the transcriptomes of 2D graphene-regulated NSC differentiation have not yet been investigated. To identify candidate genes, on which graphene may affect, we used next-generation RNA sequencing to analyze the transcriptome of NSCs differentiated for 21 days on a graphene substrate. These NSCs displayed highly enriched and differentially expressed genes compared with traditional cell culture in vitro. Of these, we identified motor protein genes that might regulate NSC differentiation, including cytoplasmic dynein and axonemal dynein genes, Ccdc108, Dnah5, and Dnah11. Furthermore, we analyzed the cell signaling pathway genes that might regulate NSC differentiation, and we constructed a protein-protein interaction network for the genes that are differentially expressed in NSCs on graphene compared to commercial tissue culture polystyrene substrates. We have identified genes potentially regulating the differentiation and migration of NSCs on graphene substrates, and our findings provide mechanistic evidence for the biological activities of graphene, especially in view of graphene-stem cell interactions.