Additive Manufacturing is noted for ease of product customization and short production run cost-effectiveness. As our global population approaches 8 billion, additive manufacturing has a future in maintaining and improving average human life expectancy for the same reasons that it has advantaged general manufacturing. In recent years, additive manufacturing has been applied to tissue engineering, regenerative medicine, and drug delivery. Additive Manufacturing combined with tissue engineering and biocompatibility studies offers future opportunities for various complex cardiovascular implants and surgeries. This paper is a comprehensive overview of current technological advancements in additive manufacturing with potential for cardiovascular application. The current limitations and prospects of the technology for cardiovascular applications are explored and evaluated.
The chondrogenic efficacy of aloe vera blended collagen-chitosan (COL-CS-AV) porous scaffold was investigated using articular chondrocytes in a standard condition. Cytocompatibility was analyzed using fluorescent dyes (calcein AM/ethidium bromide) and the viable cells were quantified by MTT assay. Glycosaminoglycan (GAG) content of ECM was estimated by using 1, 9-Dimethyl methylene Blue (DMMB). The total RNA content was quantified and the cartilage specific genes (col2a1, Acan) were amplified by reverse transcription-PCR from the cell lysate of the scaffolds. Histological examination was made using Haematoxylin and Eosin (H&E), safranin-O, masson's trichrome, alcian blue, and alizarin red to stain the specific component of ECM secreted on the construct. The cartilage specific collagen type II was estimated by immunohistochemistry using monoclonal type II collagen antibody. The results of these studies proved that COL-CS-AV scaffold has more chondrogenic efficacy than COL-CS, thus the aloe vera blend COL-CS-AV scaffold might be used as suitable candidate for cartilage tissue engineering.
In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.
Porous structure, biocompatibility and biodegradability, large surface area, and drug-loading ability are some remarkable properties of zeolite structure, making it a great possible option for bone tissue engineering. Herein, we evaluated the potential application of the ZSM-5 scaffold encapsulated GEN with high porosity structure and significant antibacterial properties. The space holder process has been employed as a new fabrication method with interconnected pores and suitable mechanical properties. In this study, for the first time, ZSM-5 scaffolds with GEN drug-loading were fabricated with the space holder method. The results showed excellent open porosity in the range of 70-78% for different GEN concentrations and appropriate mechanical properties. Apatite formation on the scaffold surface was determined with Simulation body fluid (SBF), and a new bone-like apatite layer shaping on all samples confirmed the in vitro bioactivity of ZSM-5-GEN scaffolds. Also, antibacterial properties were investigated against both gram-positive and gram-negative bacteria. The incorporation of various amounts of GEN increased the inhibition zone from 24 to 28 (for E. coli) and 26 to 37 (for S. aureus). In the culture with MG63 cells, great cell viability and high cell proliferation after 7 days of culture were determined.
Current immunological issues in bone grafting regarding the transfer of xenogeneic donor bone cells into the recipient are challenging the industry to produce safer acellular natural matrices for bone regeneration. The aim of this study was to investigate the efficacy of a novel decellularization technique for producing bovine cancellous bone scaffold and compare its physicochemical, mechanical, and biological characteristics with demineralized cancellous bone scaffold in an in-vitro study. Cancellous bone blocks were harvested from a bovine femoral head (18-24 months old) subjected to physical cleansing and chemical defatting, and further processed in two ways. Group I was subjected to demineralization, while Group II underwent decellularization through physical, chemical, and enzymatic treatments. Both were then freeze-dried, and gamma radiated, finally producing a demineralized bovine cancellous bone (DMB) scaffold and decellularized bovine cancellous bone (DCC) scaffold. Both DMB and DCC scaffolds were subjected to histological evaluation, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), fourier-transform infrared spectroscopy (FTIR), quantification of lipid, collagen, and residual nucleic acid content, and mechanical testing. The osteogenic potential was investigated through the recellularization of scaffolds with human osteoblast cell seeding and examined for cell attachment, proliferation, and mineralization by Alizarin staining and gene expression. DCC produced a complete acellular extracellular matrix (ECM) with the absence of nucleic acid content, wider pores with extensive interconnectivity and partially retaining collagen fibrils. DCC demonstrated a higher cell proliferation rate, upregulation of osteogenic differentiation markers, and substantial mineralized nodules production. Our findings suggest that the decellularization technique produced an acellular DCC scaffold with minimal damage to ECM and possesses osteogenic potential through the mechanisms of osteoconduction, osteoinduction, and osteogenesis in-vitro.
The use of gelatin microspheres (GMs) as a cell carrier has been extensively researched. One of its limitations is that it dissolves rapidly in aqueous settings, precluding its use for long-term cell propagation. This circumstance necessitates the use of crosslinking agents to circumvent the constraint. Thus, this study examines two different methods of crosslinking and their effect on the microsphere's physicochemical and cartilage tissue regeneration capacity. Crosslinking was accomplished by physical (dehydrothermal [DHT]) and natural (genipin) crosslinking of the three-dimensional (3D) GM. We begin by comparing the microstructures of the scaffolds and their long-term resistance to degradation under physiological conditions (in an isotonic solution, at 37°C, pH = 7.4). Infrared spectroscopy indicated that the gelatin structure was preserved after the crosslinking treatments. The crosslinked GM demonstrated good cell adhesion, viability, proliferation, and widespread 3D scaffold colonization when seeded with human bone marrow mesenchymal stem cells. In addition, the crosslinked microspheres enhanced chondrogenesis, as demonstrated by the data. It was discovered that crosslinked GM increased the expression of cartilage-related genes and the biosynthesis of a glycosaminoglycan-positive matrix as compared with non-crosslinked GM. In comparison, DHT-crosslinked results were significantly enhanced. To summarize, DHT treatment was found to be a superior approach for crosslinking the GM to promote better cartilage tissue regeneration.
This paper aims to investigate the dielectric properties, i.e., dielectric constant (ε'), dielectric loss factor (ε″), dielectric tangent loss (tan δ), electrical conductivity (σ), and penetration depth (Dp), of the porous nanohydroxyapatite/starch composites in the function of starch proportion, pore size, and porosity over a broad band frequency range of 5 MHz-12 GHz. The porous nanohydroxyapatite/starch composites were fabricated using different starch proportions ranging from 30 to 90 wt%. The results reveal that the dielectric properties and the microstructural features of the porous nanohydroxyapatite/starch composites can be enhanced by the increment in the starch proportion. Nevertheless, the composite with 80 wt% of starch proportion exhibit low dielectric properties (ε', ε″, tan δ, and σ) and a high penetration depth because of its highly interconnected porous microstructures. The dielectric properties of the porous nanohydroxyapatite/starch composites are highly dependent on starch proportion, average pore size, and porosity. The regression models are developed to express the dielectric properties of the porous nanohydroxyapatite/starch composites (R2 > 0.96) in the function of starch proportion, pore size, and porosity from 1 to 11 GHz. This dielectric study can facilitate the assessment of bone scaffold design in bone tissue engineering applications.
The primary role of bone tissue engineering is to reconcile the damaged bones and facilitate the speedy recovery of the injured bones. However, some of the investigated metallic implants suffer from stress-shielding, palpability, biocompatibility, etc. Consequently, the biodegradable scaffolds fabricated from polymers have gathered much attention from researchers and thus helped the tissue engineering sector by providing many alternative materials whose functionality is similar to that of natural bones. Herein, we present the fabrication and testing of a novel composite, magnesium (Mg)-doped hydroxyapatite (HAp) glazed onto polylactic acid (PLA) scaffolds where polyvinyl alcohol (PVA) used as a binder. For the composite formation, Creality Ender-3 pro High Precision 3D Printer with Shape tool 3D Technology on an FSD machine operated by Catia design software was employed. The composite has been characterized for the crystallinity (XRD), surface functionality (FTIR), morphology (FESEM), biocompatibility (hemolytic and protein absorption), and mechanical properties (stress-strain and maximum compressive strength). The powder XRD analysis confirmed the semicrystalline nature and intact structure of HAp even after doping with Mg, while FTIR studies for the successful formation of Mg-HAp/PVA@PLA composite. The FESEM provided analysis indicated for the 3D porous architecture and well-defined morphology to efficiently transport the nutrients, and the biocompatibility studies are supporting that the composite for blood compatible with the surface being suitable enough for the protein absorption. Finally, the composite's antibacterial activity (against Staphylococcus aureus and Escherichia coli) and the test of mechanical properties supported for the enhanced inhibition of active growth of microorganisms and maximum compressive strength, respectively. Based on the research outcomes of biocompatibility, antibacterial activity, and mechanical resistance, the fabricated Mg-HAp/PVA@PLA composite suits well as a promising biomaterial platform for orthopedic applications by functioning towards the open reduction internal fixation of bone fractures and internal repairs.
The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.
Recently, composite scaffolding has found many applications in hard tissue engineering due to a number of desirable features. In this present study, hydroxyapatite/bioglass (HAp/BG) nanocomposite scaffolds were prepared in different ratios using a hydrothermal approach. The aim of this research was to evaluate the adhesion, growth, viability, and osteoblast differentiation behavior of human Wharton's-jelly-derived mesenchymal stem cells (hWJMSCs) on HAp/BG in vitro as a scaffold for application in bone tissue engineering. Particle size and morphology were investigated by TEM and bioactivity was assessed and proven using SEM analysis with hWJMSCs in contact with the HAp/BG nanocomposite. Viability was evaluated using PrestoBlueTM assay and early osteoblast differentiation and mineralization behaviors were investigated by ALP activity and EDX analysis simultaneously. TEM results showed that the prepared HAp/BG nanocomposite had dimensions of less than 40 nm. The morphology of hWJMSCs showed a fibroblast-like shape, with a clear filopodia structure. The viability of hWJMSCs was highest for the HAp/BG nanocomposite with a 70:30 ratio of HAp to BG (HAp70/BG30). The in vitro biological results confirmed that HAp/BG composite was not cytotoxic. It was also observed that the biological performance of HAp70/BG30 was higher than HAp scaffold alone. In summary, HAp/BG scaffold combined with mesenchymal stem cells showed significant potential for bone repair applications in tissue engineering.
Cellulose and its forms are widely used in biomedical applications due to their biocompatibility, biodegradability and lack of cytotoxicity. It provides ample opportunities for the functionalization of supported magnetic nanohybrids (CSMNs). Because of the abundance of surface hydroxyl groups, they are surface tunable in either homogeneous or heterogeneous solvents and thus act as a substrate or template for the CSMNs' development. The present review emphasizes on the synthesis of various CSMNs, their physicomagnetic properties, and potential applications such as stimuli-responsive drug delivery systems, MRI, enzyme encapsulation, nucleic acid extraction, wound healing and tissue engineering. The impact of CSMNs on cytotoxicity, magnetic hyperthermia, and folate-conjugates is highlighted in particular, based on their structures, cell viability, and stability. Finally, the review also discussed the challenges and prospects of CSMNs' development. This review is expected to provide CSMNs' development roadmap in the context of 21st-century demands for biomedical therapeutics.
Skin injuries and in particular, chronic wounds, are one of the major prevalent medical problems, worldwide. Due to the pivotal role of angiogenesis in tissue regeneration, impaired angiogenesis can cause several complications during the wound healing process and skin regeneration. Therefore, induction or promotion of angiogenesis can be considered as a promising approach to accelerate wound healing. This article presents a comprehensive overview of current and emerging angiogenesis induction methods applied in several studies for skin regeneration, which are classified into the cell, growth factor, scaffold, and biological/chemical compound-based strategies. In addition, the advantages and disadvantages of these angiogenic strategies along with related research examples are discussed in order to demonstrate their potential in the treatment of wounds.
The polymeric composite material with desirable features can be gained by selecting suitable biopolymers with selected additives to get polymer-filler interaction. Several parameters can be modified according to the design requirements, such as chemical structure, degradation kinetics, and biopolymer composites' mechanical properties. The interfacial interactions between the biopolymer and the nanofiller have substantial control over biopolymer composites' mechanical characteristics. This review focuses on different applications of biopolymeric composites in controlled drug release, tissue engineering, and wound healing with considerable properties. The biopolymeric composite materials are required with advanced and multifunctional properties in the biomedical field and regenerative medicines with a complete analysis of routine biomaterials with enhanced biomedical engineering characteristics. Several studies in the literature on tissue engineering, drug delivery, and wound dressing have been mentioned. These results need to be reviewed for possible development and analysis, which makes an essential study.
The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes' expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.
An ideal scaffold should be biocompatible, having appropriate microstructure, excellent mechanical strength yet degrades. Chitosan exhibits most of these exceptional properties, but it is always associated with sub-optimal cytocompatibility. This study aimed to incorporate graphene oxide at wt % of 0, 2, 4, and 6 into chitosan matrix via direct blending of chitosan solution and graphene oxide, freezing, and freeze drying. Cell fixation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, alkaline phosphatase colorimetric assays were conducted to assess cell adhesion, proliferation, and early differentiation of MG63 on chitosan-graphene oxide scaffolds respectively. The presence of alkaline phosphatase, an early osteoblast differentiation marker, was further detected in chitosan-graphene oxide scaffolds using western blot. These results strongly supported that chitosan scaffolds loaded with graphene oxide at 2 wt % mediated cell adhesion, proliferation, and early differentiation due to the presence of oxygen-containing functional groups of graphene oxide. Therefore, chitosan scaffolds loaded with graphene oxide at 2 wt % showed the potential to be developed into functional bone scaffolds.
Mesenchymal stromal cells (MSCs) are widely used in the clinic because they involve fewer ethical issues and safety concerns compared to other stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). MSCs derived from umbilical cord Wharton's jelly (WJ-MSCs) have excellent proliferative potential and a faster growth rate and can retain their multipotency for more passages in vitro compared to adult MSCs from bone marrow or adipose tissue. WJ-MSCs are used clinically for repairing tissue injuries of the spinal cord, liver and heart with the aim of regenerating tissue. On the other hand, WJ-MSCs are also used clinically to ameliorate immune-mediated diseases based on their ability to modulate immune responses. In the field of tissue engineering, WJ-MSCs capable of differentiating into multiple cell lineages have been used to produce a variety of engineered tissues in vitro that can then be transplanted in vivo. This review discusses the characteristics of WJ-MSCs, the differences between WJ-MSCs and adult MSCs, clinical studies involving WJ-MSCs and future perspectives of WJ-MSC research and clinical applications. To summarize, WJ-MSCs have shown promise in treating a variety of diseases clinically. However, most clinical trials/studies reported thus far are relatively smaller in scale. The collected evidence is insufficient to support the routine use of WJ-MSC therapy in the clinic. Thus, rigorous clinical trials are needed in the future to obtain more information on WJ-MSC therapy safety and efficacy.
Joint cartilage has been a significant focus on the field of tissue engineering and regenerative medicine (TERM) since its inception in the 1980s. Represented by only one cell type, cartilage has been a simple tissue that is thought to be straightforward to deal with. After three decades, engineering cartilage has proven to be anything but easy. With the demographic shift in the distribution of world population towards ageing, it is expected that there is a growing need for more effective options for joint restoration and repair. Despite the increasing understanding of the factors governing cartilage development, there is still a lot to do to bridge the gap from bench to bedside. Dedicated methods to regenerate reliable articular cartilage that would be equivalent to the original tissue are still lacking. The use of cells, scaffolds and signalling factors has always been central to the TERM. However, without denying the importance of cells and signalling factors, the question posed in this chapter is whether the answer would come from the methods to use or not to use scaffold for cartilage TERM. This paper presents some efforts in TERM area and proposes a solution that will transpire from the ongoing attempts to understand certain aspects of cartilage development, degeneration and regeneration. While an ideal formulation for cartilage regeneration has yet to be resolved, it is felt that scaffold is still needed for cartilage TERM for years to come.
Ayurveda oil contains numerous source of biological constituents which plays an important role in reducing the pain relief caused during bone fracture. The aim of the study is to fabricate the polyurethane (PU) scaffold for bone tissue engineering added with ayurveda amla oil using electrospinning technique. Scanning Electron Microscopy (SEM) analysis showed that the fabricated nanocomposites showed reduced fiber diameter (758 ± 185.46 nm) than the pristine PU (890 ± 116.91 nm). Fourier Infrared Analysis (FTIR) revealed the existence of amla oil in the PU matrix by hydrogen bond formation. The contact angle results revealed the decreased wettability (116° ± 1.528) of the prepared nanocomposites compared to the pure PU (100° ± 0.5774). The incorporation of amla oil into the PU matrix improved the surface roughness. Further, the coagulation assay indicated that the addition of amla oil into PU delayed the blood clotting times and exhibited less toxic to red blood cells. Hence, the fabricated nanocomposites showed enhanced physicochemical and better blood compatibility parameters which may serve as a potential candidate for bone tissue engineering.
Electrospinning is a promising and versatile technique that is used to fabricate polymeric nanofibrous scaffolds for bone tissue engineering. Ideal scaffolds should be biocompatible and bioactive with appropriate surface chemistry, good mechanical properties and should mimic the natural extracellular matrix (ECM) of bone. Selection of the most appropriate material to produce a scaffold is an important step towards the construction of a tissue engineered product. Bone tissue engineering is an interdisciplinary field, where the principles of engineering are applied on bone-related biochemical reactions. Scaffolds, cells, growth factors, and their interrelation in microenvironment are the major concerns in bone tissue engineering. This review covers the latest development of biomimetic electrospun polymeric biomaterials for bone tissue engineering. It includes the brief details to bone tissue engineering along with bone structure and ideal bone scaffolds requirements. Details about various engineered materials and methodologies used for bone scaffolds development were discussed. Description of electrospinning technique and its parameters relating their fabrication, advantages, and applications in bone tissue engineering were also presented. The use of synthetic and natural polymers based electrospun nanofibrous scaffolds for bone tissue engineering and their biomineralization processes were discussed and reviewed comprehensively. Finally, we give conclusion along with perspectives and challenges of biomimetic scaffolds for bone tissue engineering based on electrospun nanofibers.
Recently, porous magnesium and its alloys are receiving great consideration as biocompatible and biodegradable scaffolds for bone tissue engineering application. However, they presented poor antibacterial performance and corrosion resistance which limited their clinical applications. In this study, Mg-Zn (MZ) scaffold containing different concentrations of tetracycline (MZ-xTC, x = 1, 5 and 10%) were fabricated by space holder technique to meet the desirable antibacterial activity and corrosion resistance properties. The MZ-TC contains total porosity of 63-65% with pore sizes in the range of 600-800 μm in order to accommodate bone cells. The MZ scaffold presented higher compressive strength and corrosion resistance compared to pure Mg scaffold. However, tetracycline incorporation has less significant effect on the mechanical and corrosion properties of the scaffolds. Moreover, MZ-xTC scaffolds drug release profiles show an initial immediate release which is followed by more stable release patterns. The bioactivity test reveals that the MZ-xTC scaffolds are capable of developing the formation of HA layers in simulated body fluid (SBF). Next, Staphylococcus aureus and Escherichia coli bacteria were utilized to assess the antimicrobial activity of the MZ-xTC scaffolds. The findings indicate that those scaffolds that incorporate a high level concentration of tetracycline are tougher against bacterial organization than MZ scaffolds. However, the MTT assay demonstrates that the MZ scaffolds containing 1 to 5% tetracycline are more effective to sustain cell viability, whereas MZ-10TC shows some toxicity. The alkaline phosphatase (ALP) activity of the MZ-(1-5)TC was considerably higher than that of MZ-10TC on the 3 and 7 days, implying higher osteoblastic differentiation. All the findings suggest that the MZ-xTC scaffolds containing 1 to 5% tetracycline is a promising candidate for bone tissue healing due to excellent antibacterial activity and biocompatibility.