Additive Manufacturing of Biopolymers for Tissue Engineering and Regenerative Medicine: An Overview, Potential Applications, Advancements, and Trends

Abstract

As a technique of producing fabric engineering scaffolds, three-dimensional (3D) printing has tremendous possibilities. 3D printing applications are restricted to a wide range of biomaterials in the field of regenerative medicine and tissue engineering. Due to their biocompatibility, bioactiveness, and biodegradability, biopolymers such as collagen, alginate, silk fibroin, chitosan, alginate, cellulose, and starch are used in a variety of fields, including the food, biomedical, regeneration, agriculture, packaging, and pharmaceutical industries. The benefits of producing 3D-printed scaffolds are many, including the capacity to produce complicated geometries, porosity, and multicell coculture and to take growth factors into account. In particular, the additional production of biopolymers offers new options to produce 3D structures and materials with specialised patterns and properties. In the realm of tissue engineering and regenerative medicine (TERM), important progress has been accomplished; now, several state-of-the-art techniques are used to produce porous scaffolds for organ or tissue regeneration to be suited for tissue technology. Natural biopolymeric materials are often better suited for designing and manufacturing healing equipment than temporary implants and tissue regeneration materials owing to its appropriate properties and biocompatibility. The review focuses on the additive manufacturing of biopolymers with significant changes, advancements, trends, and developments in regenerative medicine and tissue engineering with potential applications.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

The various aspects such as type of tissue and the hormones necessary for the discrepancy and physical size are restricted to this regeneration as the body can regenerate amazingly (critical defect). Any tissue damage beyond this crucial dimension requires external assistance approaches such as tissue engineering (TE) and regenerative medicine (RM), in which the external hollows are termed yardsticks. These tissues offer a platform for cellular activity and new tissue creation [1]. In TE and RM, the scaffolds have a crucial role. These tissues are frequently supplied with growth agents in order to accelerate the differentiation between cells and selected lines to encourage the development of new tissue. For cell viability and cell prolife, the physical and chemical content plays a crucial part [2]. Biomaterial is classified according to a wide variety of parameters, including chemical and physical composition, biodegradability, origin, and modification generations. Biomaterial is selected depending on the target tissue. Biomaterials are divided into ceramics, polymers, and composites based on their chemical composition. The biomaterial class of ceramics includes important components of inorganic metal or calcium salts [3]. The primary usage of these biomaterials has been orthodontal. Because of its resemblance with binding tissues, polymers are employed in soft tissue engineering. Mixes of ceramics and polymers comprise the composite class of biomaterials. The composites have orthopaedic and dental TE uses. Natural and manmade polymers of TE and RM are commonly employed [4]. The biodegradability and biocompatibility in natural biomaterials have plentiful availability such as collagen, chitosan, hyaluronic acid, and alginate that are commonly employed. One of the key aspects of natural polymers is the degradation of biomaterials [5].

Since these biomatters are present in the extracellular matrix (ECM), cells are very compatible and respond to growth. Collagen is one of the most frequently used natural biomaterials in several applications of scaffolds. Biopolymers have recently gathered significant interest with a view to biocomposites with a multifunctional and high efficiency that have a low environmental effect, with exclusive accessibility, renewable, environmentally friendly, and lightweight qualities [6]. Biopolymeric composites should replace for the multifaceted application of synthetic materials in optics, biochemistry, and biomedical engineering [7]. The product data is divided into two dimensions. The data are sent through the machine from the basis of the product layer by layer, and the material is dropped layer by layer, which in an additive process infuses the newest layer of material into the old layer as shown in Figure 1. The researchers have received tremendous attention in recent years from biopolymers and biodegradable synthetic polymers. Biomedical applications require the production of sustainable, stronger, and lightweight biopolymeric materials [8]. The development or choice of ways to tackle the issues of architectural design however needs a compromise between visions and aims, which generally conflicts with new biomaterials [9].

[figure omitted; refer to PDF]

However, bone tissue engineering is regarded as an alternative in situations when donor availability is restricted, or where there is a risk of disease transmission, donor site difficulties, or even limitations of external materials to reshape and respond to physiological conditions. This is true whether the scaffold is acellular or seeded with stem cells, which can directly develop into bone cells, to replace a broken portion of bone. The scaffold’s composition and structure are critical. Bone tissue engineering’s primary goal is to create scaffolds that not only act as a scaffolding for the implantation of cells but also send regenerative signals to cells to accelerate bone healing and repair. Structural bone scaffolds are 3D architectures and environments that are designed to (1) promote cell adhesion and survival, (2) accelerate bone remodeling and remodeling, (3) provide osteoconductive structural guidance, and (4) in some cases, act as carriers for growth factors, antibiotics, or gene therapy. The epidermis which works as an anti-illness shield is the most waterproof layer and plays a significant role in bodily temperature and humidity regulation. More than 90 percent of epidermal cells are keratinocytes [137]. Langerhans, melanocytes, and Merkel cells dominate the bulk of epidermal cell populations. The dermis and skin base are around 90 percent of the skin’s weight. It is an extracellular matrix of soft tissues consisting of a variety of cells, lenses, and hair follicles. The dermis has a strong vascularization, and the nerve ends with a blood vessel [138]. Fibroblast is the largest dermal cell containing collagen and elastin and giving mechanical strength to the skin. A more deeply elastic, mucous tissue-cell skin that store fat, blood vessels, and nerve is present in the pulmonary hypoderm. Traumas such as physical penetration, venom, fire, illness, and operation are the major reason and contribute to the chance that important organs are infected, injured, or dehydrated by this disease [139]. Skin replacement technology offers a potential foundation for better care for combating chronic and acute skin damage. However, given the mechanical and physiological aspects of active skin, cellular basis technology and simulated extracellular matrix are required for skin tissue engineering to connect with the surrounding tissue [140, 141]. No substantial skin prototype is currently available to accurately replicate the natural skin structure, composition, organic consistency, or visual environment. Alternatives of the skin might have crucial, easy-to-use, and wound-specific characteristics [142].

These biomaterials are sufficiently water-sensitive and have specific affinity to host places. Their biochemical and mechanical qualities are sufficient, their privation is controlled, their disinfection is nontoxic and nonantigenic, and their inflammation is minor [143]. They can also join the congregation at low operational cost with minimum injury and suffering of angiogenesis. The ultimate objective of tissue technology is to achieve the maximum of these needs to prepare intelligent skin substitutes [144]. In addition, the new skin electronic properties or aesthetic structure do not restore polymeric composite materials. In order to extend skin growth to provide the typical usefulness and beauty of healthy skin, the changes in stem cell biology and skin morphogenesis are necessary [145]. Some of the biopolymeric materials and their features are represented in Table 2.

Table 2

Biopolymeric materials in tissue engineering and wound healing [146–155].

Biomedical field	S. No.	Polymeric material	Prominent characteristics
Tissue engineering	1	Chitosan	Recyclable, biocompatible, uncontaminated
	2	Gelatin	Bioactive, biocompatible, hemocompatible, cell adherence
	3	Arabinoxylan	Biocompatible, uncontaminated, cell observance, bioactive, cell explosion
	4	Collagen	Biodegradable, fibrous, biodegradable, cell proliferation
	5	Xyloglucan	Cell explosion, environmental, cell discrepancy, biocompatible
	6	Fibrinogen	Biocompatible, hemocompatibility, cell propagation, decomposable
Wound healing	7	Arabinoxylan/guar gum/gelatin/collagen	Antiseptic, biocompatible, decomposable, bioactive, continuous drug release, cell propagation
	8	Chitosan	Biocompatible, antibacterial, cell proliferation, bioactive
	9	Alginate/fibrinogen/hyaluronic acid/xyloglucan	Fiber protein, biocompatible, recyclable, rubbery, sterile, cell obedience
	10	Bacterial cellulose/pectin	Antibacterial, cell adherence, cell differentiation, biocompatible, bioactive, cytocompatible

6.3. Wound Healing

Wounds are a form of uneven skin punching, breakdown, or skin deformation owing to a chronic or thermal trauma. Injuries can be classed as chronic or acute injuries depending on the healing procedure. Chronic injury is predominantly tissue lesions, usually within 8 to 12 weeks, which appear to have totally resolved [156]. Acute injuries continue to occur and are still more than 12 weeks of recuperation. Various neurological factors can lead to wound-healing impairments or the failure to correctly heal. Chronic injury examples are bedsores and leg ulcers. As the basis for the wound gradation, skin layers and polluted areas are used and only the epidermal skin surface is involved with surface wounds [157, 158]. The word partial thickness injury is defined as injury involving the epidermis, deep epidermis, muscles, soft tissue, and follicular tissues. The wounds are combined with subcutaneous fat or deep tissues besides the epidermis and the skin surface [159]. The physiological wound repair is part of coordinated teamwork among various biological systems. The wound is entirely treated in a cascade with controlled operations. Hysteresis and coagulation of your blood start with lesions, mainly in order to avoid first sight exsanguinations, taking place in every area of the body [160]. The lesion is also a long-term secondary target and a matrix for cell adherence. A carefully managed balance of endothelial cells and thrombocytes relies on the homeostasis and fibrin produced at a site of the injury [161]. The neurological system of response in damaged veins causes vascularization, which blocks blood flow over several minutes. The waterfall of coagulation is caused by homeostatic behaviours and proliferation and differentiation [162]. Platelets bond when blood spills, causing a release of the coagulation factor: fibronectin, fibrin, vitronectin, and thrombospondin. Coagulation retains homosexuality and a cell migration matrix in homosexual and inflammatory treatments [163, 164]. Many biopolymers are routinely used in wound care and treatment, including fibrous proteins and different polysaccharides. These biocompatible, biodegradable polymer matrices preserve an atmosphere similar to the extracellular environment. The process of sluggish wound treatment is accelerated [165]. For cell adhesion, proliferation, migration, and differentiation, the biopolymeric matrix provides an ideal microenvironment Using biopolymer-based wound care materials, three-dimensional cross-linked polymeric networks can keep the wound wet and oxygenated. As a result of the use of wound healing dressings, the wound is regenerated, prevented, and protected from disease-causing bacteria. Dermal and epidermal tissue healing and regeneration rely on it. This wound healing material is identified as hydrogels that can be packed for localised therapeutic delivery with spatially and temporally controlled cells, medications, and peptides. Hydrogels have been utilised for biomedical and therapeutic applications, such as tissue engineering, regenerative medicine, cancer treatment and infectious diseases, controlled drug delivery, and peptide delivery [166]. Hydrogels adhere to the application site shape to provide for considerably more therapeutically practical formulation of loaded hydrogels in biomedical applications. While hydrogels are believed to be exceptionally biocompatible with poly(ethyleneglycol) (PEG), hydrogels based on PEG, hydrogels used on are considered extremely biocompatible. High systemic biocompatibility of PEG and utilisation of biomaterials generated from ECM increase the distribution of cell growth [167]. As a result, the multifunctional wound-care material PEG-based cross-linking hydrogels with good loading components, such as cells, medicines, and peptides, are being developed [168].

6.4. Bioprinting

Bioprinting includes production of AMs in complex and functioning living tissues, utilising biocompatible cells, supported components and materials. In regenerative medicine, biopharmaceutical products are generally used to support tissue and organ transplant, especially the development of hydrogel [169]. Many forms of bioprinting are available, including inkjet printing and AM-based extrusion. One study generated 3D cell architectures through neural cell sheets employing an alternative human pluripotent embryonal carcinoma (NT-2) cells and fibrin gel inkjet printing approach [170]. The Vascular Endothelial Growth Factor (VEGF) presence in 3D-bioprinted scaffolds that incorporates alginate into one of their matrix mixes promotes vascularization in gelatine microparticles.

The hydrogels developed containing hyaluronic acid and semi-interpenetrating systems with a dextran basis [171]. The use of neural stem cells to produce artificial neural tissue was organically printed with collagen and VEGF-releasing fibrin gel. Hyaluronic acid-based scaffolds were created through layer-by-layer deposition through bioprinting [7]. In order to print bespoke steaks with cell inclusion, many such techniques have integrated other conjoined natural polymers such as dextrin and gelatin. This has led to the development of sophisticated materials with biological activity by adding growth factors such as Bone Morphogenetic Proteins (BMP-2) by use of microfabrication technologies [172]. Many of the bioprinting approaches mentioned could be adjusted and optimised with or without cell utilisation for bone tissue engineering. The survivability of cells in situ following the printing method is part of many issues related to cell printing [173]. New methods used for obtaining 3D cell-charged structures with proper mechanical and biological properties have been applied with collagen-based bioinks [174]. The 3D printing techniques for polymers are shown in Figure 6.

[figure omitted; refer to PDF]

6.5. Advanced Functional Biomaterials

In order to design and synthesise multifunctional polymer material, a better understanding of the sequence, structural, and functional features of natural polymers plays an essential role. These innovative artificial biomaterials are self-assembled and stimulated to encourage cell contact and growth under particular conditions [176, 177]. The complexity of posttranscriptional changes has limited sophisticated and multifunctional biomaterial protein synthesis that utilises bacterial resources and the conundrum of target genes [178]. Other changes have been intended to properly control spatial and temporal releases. The development of the structure and de novo design for protein-based biomaterials has been made easier by progression in gene therapy and manipulation approaches [179]. Due to the existence of multifunctional domains on the protein structure, the structure of produced biomaterials is linked to significant versatility, such as cell binding places and enzymatic domains. The design and production of new biomaterials based on artificial proteins have been promised recently in genetic engineering. Compared with its native counterparts, these biomaterials have a unique performance, such as improving self-assembly in fiber architectures [180, 181]. The significant necessities for choosing a bioink for 3D printing in biomaterial characteristics is shown in Figure 7.

[figure omitted; refer to PDF]

6.6. Materials and Manufacturing Advances and Trends

The selection of optimum biomaterials will be a vital part of effective bioprinting of therapeutically relevant tissue. Based on the availability and knowledge of these materials, numerous polymers were examined during the bioprinting stage for traditional 3D printing and fabric production [183]. However, in bioprint applications, materials are not the most physiologically suitable. Many of these are exceedingly physiologically active, leading to improper cell contact and premature or undesired differentiation of the stem cells [184]. The focus is currently on new biopolymers and hydrogels, which imitate better the nanostructural characteristics and reactivity of ECM and other constituents in the true tissue microenvironment. But those new hydrogels and biopolymers more biocompatible are not necessarily appropriate to conventional methods of bioprinting [185]. Many lack the structural stability to optimise bioprinting and can collapse if they are too soft. An interesting field of research is to optimise the microarchitecture for these biopolymers. Substances are combined with the proliferative and cytocompatible impact of a softer material to optimise the usefulness of all of them, the mechanical properties of one single substance [186, 187]. For example, an “integrated tissue organ printer” is utilised to put companies into the soft hydrogel cell scaffold. Tricalcium phosphates with gelatine and hyaluronic acid bioprinting can be successfully combined [188]. In general, the effectiveness of the bioprinting process has to be enhanced. The existing bioprinting method is time consuming and currently cannot reliably supply the number of cells needed for varied tissue types.

As mentioned before, a change in cell shape, changed signalling pathways, and even cell death is often caused by imposed force through the printing process [189]. In order to make more efficient cell death and loss, the huge effort is involved in each bioprinting project. Improved methods for monitoring and assessing cell death are part of the solution. Vascular networks may be the main task in converting bioprinting into the lab for the production of functional tissues [190]. Tissues of even minimal complexity will not survive without proper channels for nutrition delivery and waste removal. In vivo, the diffusion of oxygen is limited by a vascular network for tissues that are beyond 100-200 mm. Infected tissues will have nutritional restrictions without a vascular network which result in inadequate development of tissue or necrosis [191]. In order to properly perfect bioprinted tissue, an early enough developmental network must be established for the prevention of death of tissue and for the endothelium to be attached and grown. As a result of the development process, all tasks in normal development must be played by the vascular structures, including the maintenance of selective waste and nutrient barriers, and inflammatory reactions, coagulation, and other homeostatic processes [192, 193]. Today, problems with bioprints are mostly related to restrictions on printing resolution and speed. Capillaries, for instance, can have a diameter of about 3 mm while a droplet of 20 mm is currently used by the highest-resolution laser-based bioprinters.

Conventional methods or additive manufacturing can be used to create bone scaffolds. Pore size, shape, distribution, and interconnectedness of pores are all difficult to manage using conventional approaches. To add living cells in conventional procedures would be very impossible because of manufacturing circumstances. If pores are distributed in an unintended manner, it could have a negative impact on cell distribution and, ultimately, the development of new tissue. Other organic solvents left behind in the scaffold microstructure can negatively affect cell survival or function. Due to low-cost items and simple instrumentation, these techniques are still employed today [194]. As a result of the absence of hazardous solvents in AM procedures, the biocompatibility of scaffolds is much improved as well. If necessary, scaffolds can be constructed with two or more materials. Despite the high resolution of SLA and SLS, their applicability in the manufacture of bone scaffolds is extremely limited. Photosensitive polymers required for SLA use in bone tissue have a low biocompatibility. As a result of the high-intensity laser beam, SLS is not generally used in bone tissue applications. In spite of its low resolution and limited material options, FDM solvent-free and ultraclean procedure is likely to be the greatest technology for incorporating live cells, which could explain why FDM-created PCL bone scaffolds have won FDA approval.

Even if printing resolution is increased to such an extent that a complex capillary network can be produced, time with the currently available technology is prohibitive [195]. The cell viability may be impacted if the printing cannot be finalised fast. In consideration of these issues, several solutions have been proposed. One of the most promising is attempts to vascularize in vivo with the addition of angiogenic substances to biomedical tissue implants, inducing the host vasculature growth. This method has to be refined, despite encouraging outcomes [196, 197]. Alternatively, vascular networks of synthetic origin have been attempted. While the bioprinting of vessels with bigger diameters has been successful, synthetically created small microvascular grafts with fewer than 5 mm show poor patentability and are now unrealistic [198]. Inappropriately, the basic problem of tissue death prevention and timely growth of mature, functional vasculature has still to be overcome [199].

6.7. Challenges and Future Directions

Two types of tissue engineering difficulties exist: novel bioink research and development for specific tissues or universal bioink for all tissues and the regulatory category. Ideally, a universal bioink must be a biomaterial mix that promotes survival in the angiogenesis and in nerve intercalation of natural tissues, chemical indicators, and growth factors. These challenges can be overcome by providing new technologies, such as additive fabrication, which allow the production of complicated fabrics. Vascularization is one of the most essential difficulties for developing sustainable angiogenesis solutions involving the addition of angiogenic growth factors, platelet additions, bone marrow clots, and bioreactors. Since numerous heads loaded with cell type can be used by bioprinters, a vasculature is placed into a 3D imprint. The use of sacrificial biomaterials within the skin is another technique to address vascularization. Sacrificial materials provide mechanical support throughout the construction of the 3D printing process. During the postprocessing, processing of the buildings from the channels or empty regions in the building can be quickly dissolved or removed as circulatory channels.

Graphene and their composites and metal nanoparticles have also taken on a crucial importance as fillers into biopolymers reinforced their mechanical characteristics, such as tensile, effect, bending, and other structural qualities in medical applications, to create the necessary biomaterials. The main issues of the usage of biopolymers for synthesising biocomposites are mechanical behaviours and inadequate dispersion. The fillers produce agglomerates with a matrix of biopolymers that leads to feeble interfacial connection with defective structural harmonics and imperfect mechanical characteristics. The outcome is a large number of additional unusual properties, such as susceptibility to high temperatures, humidity, low impact strength, shelf life, and more. Future guidelines lead to new biomaterial in order to meet the above concerns and to be economically viable, recycled, and eco-friendly.

7. Conclusion

The biopolymers are the greatest option for synthetic petroleum polymers with considerably renewable, biodegradable, and environmentally sound characteristics. Biopolymers are not supported by mechanical properties such as high strength of tensile, impact strength, bending force, and thermal stability. However, they are able to perform load-bearing applications using their ceramic composites using a mechanical strength. There is yet more attention to be paid, inventions and improvements by using reinforced elements to adapt biocomposite microstructural features, the standard mixing techniques.

(i) These composites lead to many other unusual qualities such as sensitivity to high temperatures, susceptibility to moisture, low impact, and shelf life

(ii) In order to address the specified factors and to fit economic viability, recyclability, and eco-friendly ways, future direction leads to new biomaterials. This combination of synthetic and natural macromolecular chemistry leads essentially to biomedical applications since polymer structure management can lead to functionality being manipulated

(iii) Bioprinters can automate the assembly process and permit the preprogram and intricate manipulation of biopolymers, from macromolecular to the live cell. This is done to achieve architectural and biochemical complexity which is never previously achievable especially in biomedical fields of tissue engineering and regenerative medicine

(iv) Tissue engineering has generated both natural and synthetic polymers through the technique of 3D printing, and various other materials have been developed. In combination with polymers, fiber and particles are developed to produce materials with enhanced bioactivity, biocompatibility, and physical and chemical qualities

Acknowledgments

The authors thank Chennai Institute of Technology, Chennai, and Saveetha School of Engineering, SIMATS, Chennai, for the technical assistance. The authors appreciate the supports from Wollo University, Kombolcha Institute of Technology, Ethiopia. This research was performed as a part of the employment of Wollo University, Kombolcha Institute of Technology, Ethiopia.

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