Use of Natural Biomaterials in Tissue Regeneration

2.1 Polysaccharide-based biopolymers

Polysaccharides, the biosphere’s most abundant macromolecules, are produced from glycoside bonds. They are an important component of plant and animal skeletal structures. Polysaccharides exhibit remarkable structural diversity compared to proteins due to the numerous combinations of sugar isomers and chemical bonds. This allows them to influence cellular environments. Natural polysaccharides show challenges for scaffold fabrication. The difference in their distribution, branching, and sequence of molecular weight hinders biological interactions and material flow, and many lack biodegradability. Therefore, chemical modifications of polysaccharides are required for biomedical applications. Chitosan, alginate, hyaluronic acid (HA), pectin, cellulose, and agarose are classified as the most common polysaccharides. Table 1 introduces and summarizes the properties of common natural biopolymers in tissue engineering and regeneration [4, 23, 27, 28].

2.2 Polypeptide- and protein-based biopolymers

Proteins and peptides are agro-polymers, which can be derived renewably from animals, plants, and microorganisms. Peptides are shorter polymeric chains with ≤100 amino acids, while proteins have longer polymeric chains with ≥100 amino acids. Protein-based biopolymers utilized in tissue engineering include collagen, soybean proteins, fibrin, silk fibroin (SF), gelatin, etc. [29, 30]. Protein-based polymers are made up of amino acids bound together by strong amide bonds. They have acceptable biocompatibility and the ability to cause desired tissue responses, which make them ideal for tissue engineering. However, their usage in biomedical implants is limited by processing issues, possible immunological reactions, and insufficient mechanical strength, especially for load-bearing applications such as bone scaffolds [4, 13, 31]. Figure 1 illustrates various types of natural biopolymers used in tissue engineering and regenerative medicine.

3.1 Skin tissue regeneration

The skin—the body’s first line of defense—is a complex organ consisting of the epidermis and dermis [33]. They can be deep or shallow, acute (burns, surgical incisions) or chronic (diabetic wounds, bedsores). Acute wounds typically heal within a couple of months, but chronic wounds take longer to recover from as they are often impacted by variables, including infection and inflammation, or underlying health conditions that often impact deeper tissues [34, 35]. Wound healing is a highly organized process that involves multiple cell types, growth factors, cytokines, and ECM components and occurs in four overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Wound healing is a complex process involving a cascade of cellular events. The first phase is hemostasis, in which a blood clot forms, and the second phase is inflammation, when immune cells (neutrophils and macrophages) invade the area to remove debris and release growth factors [35]. During the proliferative phase, keratinocytes move to reepithelialize the wound, fibroblasts produce new ECM (mostly collagen type III), and new blood vessels grow (angiogenesis). Hair follicles and sweat gland epithelial stem cells also help to reepithelialize the skin. Activation of key signaling ways, like Wnt/β-catenin, hedgehog, and notch, is critical at this phase. Finally, during remodeling, the ECM is reformed (collagen type III is replaced by stronger collagen type I), excess cells are eliminated, and the wound compresses to its final shape [36, 37, 38].

The successful completion of each healing step depends on a complex interaction of signaling molecules. Growth factors (epidermal growth factor (EGF), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGF-β)) and cytokines (interleukin (IL)-1β, -6, -8, -10, and tumor necrosis factor alpha (TNF-α)) help regulate cell activity and coordinate healing processes [38]. TGF-β stimulates fibroblasts and affects ECM synthesis, while interleukins regulate inflammation and immunological responses [39]. The body has an extraordinary natural healing mechanism, but the molecular mechanisms that regulate postnatal wound healing are different from those controlling embryonic skin growth. Therefore, it inhibits complete regeneration and frequently leads to scar formation [4]. Further research into these pathways, as well as the creation of biomaterials and medicines that resemble embryonic healing, may lead to improved wound management and tissue regeneration.

Currently, different skin treatments, such as ointments, wound dressings, and medical devices, are used; however, effective skin regeneration and wound healing remain a significant medical challenge. Gauze as a traditional dressing can be painful due to adherence to the lesion. Modern dressings make some improvements, but actual tissue regeneration is necessary. Thus, there’s a continued need for wound healing, and tissue regeneration using biomaterials is a key focus of global research. Various natural polymers, including chitosan, collagen, HA, alginate, and silk fibroin (SF), are used to promote wound healing and skin regeneration [40].

Chitosan improves wound healing and polymorphonucleated leukocyte (PMN) migration by increasing IL-8 excretion from neutrophils. Neutrophils overly express IL-8 (a neutrophil chemoattractant) in response to chitosan. This expression correlates with the degree of chitosan acetylation and affects hydrophobicity and PMN interaction [41]. In addition, chitosan improves macrophage activity during tissue regeneration, including inflammation, antigen presentation, phagocytosis, cytokine production (TNF-α, IL-1β, nitric oxide (NO)), and growth factor release [42]. Calcium alginate has demonstrated positive effects on skin wound repair and collagen synthesis. Now, commercial alginate-based substrates for wound dressing are available. Alginate helps diabetic wounds heal faster by increasing the ratio of collagen types I/III. The addition of drugs like Simvastatin improves angiogenesis through Akt/Erk signaling and upregulating hypoxia-inducible factor 1-alpha (HIF-1α) and VEGF [43, 44, 45].

SF is an attractive biopolymer used in wound healing applications. It improves cell proliferation, differentiation, and adhesion, accelerating healing through nuclear factor-kappa B (NF-κB) signaling. SF increases fibroblast activity, which is crucial for skin tissue repair. Produced peptides following trypsin SF digestion enhanced fibroblast proliferation. The addition of collagen to SF increases fibroblast proliferation. As a result, SF’s biocompatibility, affordability, hemostatic properties, ability to improve cell migration/recruitment, exudate absorption, and favorable mechanical properties make it widely applicable in wound care. Keratin, as another biopolymer, has been demonstrated to accelerate the process of wound repair [46, 47].

3.2 Bone tissue regeneration

More than 75 million people worldwide suffer from bone-related disorders, including bone fractures, osteoporosis, and bone defects. Traditional therapies like autografts, allografts, ceramic-coated, or inert metallic implants are quite expensive. Autologous bone is an efficient bone regeneration approach that promotes bone growth by direct bone bonding (osteoconduction) and differentiation of stem cells into bone cells (osteoinduction). This method elicits no immune reactions [1, 48]. Every year, grafted bones present a number of obstacles, including shortages of products, donor complications, and a 50% failure rate [47]. Biomaterial scaffolds are an excellent technique for bone tissue regeneration due to their osteoinduction and osteoconduction capabilities. Natural polymers, such as collagen, chitosan, alginate, and SF, are commonly used in bone tissue engineering due to their cost-effectiveness, biocompatibility, degradability, adaptability, and desirable mechanical qualities [49, 50]. Bone fractures and osteoporosis are leading causes of disability. While fractures commonly repair spontaneously, total restoration of bone strength and structure is not always possible. Fracture healing continues via inflammation, repair, and remodeling phases, with a strong emphasis on osteogenesis (the generation of new bone) supported by osteoprogenitor cell proliferation and osteoblast maturation. Drug therapy alone is frequently insufficient for full regeneration, emphasizing the necessity for biomaterials that stimulate osteogenesis to adequately restore fractures and bone deformities [4, 51]. Fracture healing and bone formation occur following complex molecular and cellular functions, particularly the expression of genes required for osteoblast differentiation. Runx2, a transcription factor, is critical for osteoblast development and mineralization [4]. Alkaline phosphatase (ALP) and osteocalcin (OCN) are two recognized osteoblast biomarkers that control osteoblast function and ECM mineralization during osteogenesis and restoration of bone. The increased expression of these biomarkers in osteoblasts indicates cell differentiation and maturation. Natural polymers, owing to their biocompatibility and biodegradability, are preferred compared to synthetic polymers in bone regeneration because they provide customized structures that can connect to cell receptors and serve as sites for enzymatic degradation [4, 52].

SF shows an extraordinary capacity for bone repair due to its structural similarity to collagen type I. Chitosan, with its antibacterial properties, shows promise in bone restoration. However, due to its lack of inherent osteoconductivity, chitosan is often combined with materials like hydroxyapatite (HAp) or calcium phosphate in composite scaffolds to better mimic bone. Chitosan nanofibers, when activated with Runx2 mRNA and protein, have been shown to increase osteoblast mineralization, proliferation, and maturation through Runx2-mediated regulation of osteoblast-associated gene expression [52]. Cellulose-based materials are considered a viable material for bone tissue regeneration because of their structure, high crystallinity, and high-water absorption capacity. Furthermore, the addition of particles in biopolymers increases mechanical properties and allows for better mimicry on bone [1].

Biomineralization, using simulated body fluid (SBF), is a valuable technique to induce apatite formation on porous scaffolds. It is performed for developing nanofeatured structures and predicting in vivo bone formation. Mineralization on biopolymers is enhanced by charged pendant groups that promote apatite nucleation. Functional groups with negative charges (like carboxylate and phosphate) and nucleation agents (like calcium phosphates and anionic proteins) are commonly used to electrostatically induce mineralization that leads to various biopolymer/calcium phosphate scaffolds for bone regeneration [53].

HAp, a stable calcium phosphate that resembles bone, is a common bioceramic in polymer matrices because of its osteoconductive properties, biocompatibility, minimal immunogenicity, and capacity to bind to hard tissue. HAp, which is made from phosphates found in biological fluids at a physiological pH, is appropriate for bone allografts and metal implantation [54, 55]. Despite rising valuable research on bone tissue creation, a lack of clinical proof impedes commercial applicability. To achieve this goal, more detailed research is needed to understand bone repair stages and design effective biopolymers for bone tissue regeneration.

3.3 Cartilage tissue regeneration

Cartilage tissue regeneration is a long-standing challenge in regenerative medicine. Autologous grafting is a common therapy method. The American Academy of Orthopedic Surgeons (AAOS) reports that over 6 million people in the US suffer from knee-related disorders annually [1]. The damaged cartilage displays a low capacity for self-healing due to limited access to lymph nodes, neurons, blood vessels, and chondrocytes, which has limited migration potential. Medication can positively influence osteoarthritic injuries and cartilage regeneration [55]. Additionally, anterior cruciate ligament (ACL) and osteochondral defects are other types of cartilage disorders that are predicted to cause disability in the near future. Postsurgical cartilage regeneration is challenging due to cartilage’s slow metabolism, creating a need for nonsurgical solutions. Cartilage, which protects bones in joints, comes in three types (fibro, hyaline, and elastic), with articular (hyaline) cartilage being essential for load bearing. This hyaline cartilage has a unique ECM made up of collagen and proteoglycans, which contributes to its flexibility and wear resistance [56]. Hyaluronan, heterotypic collagen fibrils, and proteoglycan glycosaminoglycan webs of aggrecan are the primary compounds of cartilage ECM, which serves as a wear-resistant and flexible tissue. Cartilages in the larynx, ear, and epiglottis are more flexible than hyaline. The fibrocartilage in the knee and between the vertebrae is rigid. Less blood vessels and neurites cause limited cartilage regeneration, leading to rheumatoid arthritis, inflammatory illness, and joint degradation. Cartilage injuries cause ECM degradation and the recruitment of joint chondrocytes from adjacent locations, as well as decreased filtration and inflammatory cell vascularization [57, 58].

Various types of biopolymers, including collagen, fibrin, gelatin, cellulose, agarose, alginate, chitosan, and elastin, are utilized for cartilage tissue engineering. Several factors, such as growth factors and cytokines (transforming growth factors β (TGF-βs), platelet-derived growth factors (PDGFs), insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), parathyroid hormone-related protein (PTHP), Wnt signaling pathway, etc.), and nutritional and metabolic factors (e.g., glucose, amino acids, and vitamins), are involved in chondrogenesis. TGF-βs promote chondrocyte proliferation, differentiation, and upregulation of chondrogenic transcription factors, resulting in collagen type II and aggrecan production [58, 59, 60].

When biopolymers fill the injured site, they promote cartilage regeneration by showing minimal donor site trouble, fewer implantation obstacles, and faster healing. To create successful cartilage-based tissue building, appropriate biomaterials must be used. These biopolymers should generate two distinct phases of connective tissue, a biomimetic area, and local cartilage frameworks that integrate with the native tissues. Collagen type II and glycosaminoglycan (GAG) are essential for the maintenance of chondrocytic phenotypes and stimulating chondrogenesis. Without them, chondrocytes change and produce fibrocartilaginous structures enriched in collagen type I, resulting in hyaline formation failure [4]. In a study, a chitosan-fabricated scaffold combined with rat mesenchymal stem cells (MSCs) showed regeneration of cartilage tissue. This three-dimensional (3D) matrix of scaffold protects MSCs in the injured area and reduces poor transplanted cell distribution, which could be a limiting factor for the efficacy of autologous cell implantation [61]. Furthermore, an in vivo investigation found that chitosan-based hybrid scaffolds with HA promote chondrogenesis [62]. Another study demonstrated the healing properties of a chondrocyte fibrin solution, which resulted in efficient deposition of cartilage-specific ECM components [63]. Lately, numerous studies have been conducted on cartilage tissue repair and regeneration. However, the development and evaluation of scaffolds present a number of obstacles, including biomaterial selection, manufacturing procedures, and application. Their biological stability, biocompatibility, mechanical qualities, adequate biodegradation of produced scaffolds, and drug/growth factor administration are critical for cartilage repair and regeneration.

3.4 Cardiac tissue regeneration

The heart is a muscular organ that pumps and circulates blood through the circulatory system [64]. Poor regeneration and repair occurred in mature humans after heart injury or cardiac failure. Apoptosis (automated cell death), necrosis (injury-caused cell death), and oncosis (ischemic cell death) can all harm cardiomyocytes (CMs), resulting in cardiac dysfunction. Pathological conditions of the heart include necrosis and apoptosis. In oncosis, cells swell instead of shrinking, leading to damage. Myocardial infarctions during cardiac pathogenesis cause scar tissues and CMs sections are exchanged for fibrillar collagen and fibroblast-like cells. Cardiac tissue engineering has not advanced to the clinical level; however, stem cell transplantation has shown promising results in clinical cardiac trials. Cardiovascular diseases are strongly linked to increased oxidative damage to vascular endothelial cells. Reactive oxygen species (ROS) generation by vascular endothelial cells has a significant impact on the progression of various essential clinical illnesses, including atherosclerosis and hypocholesterolemia. High ROS levels in cells cause irreversible harm in a variety of ways, including the inactivation of key enzymes, membrane lipid oxidation, and apoptosis initiation. As a result, materials that may reduce the number of free radicals can prevent oxidative stress of cells. Researchers have focused their attention on polymer-based scaffolds transplanted with stem cells and external stimulation to repair damaged cardiac tissues [65, 66]. Biopolymers like collagen, chitosan, alginate, gelatin, fibrin, etc., are widely applied for cardiac tissue engineering. Chitosan can prevent damage from oxidative stress. Studies show that it lowers ROS levels similar to natural antioxidants and stops lipid oxidation. It also changes how endothelial cells release cytokines. Chitosan oligosaccharide reduces IL-8 production (by blocking certain signaling pathways) and lowers intercellular adhesion molecule 1 (ICAM-1) and E-selectin expression (by affecting mitogen-activated protein kinase (MAPK) and NF-κB) [67, 68].

Mimicking tissue is critical in tissue engineering and regeneration; thus, substrates like hydrogels and scaffolds must be elastic and conductive in cardiac tissue engineering. Also, minimally invasive ways should be performed to decrease the risks and expenses associated with surgical procedures in tissue engineering. In research, collagen, Matrigel, and fibrin glue were utilized as thermosensitive injectable biopolymers, which increased angiogenesis and myofibroblast influx. Furthermore, another study found that injectable alginate-based hydrogels might cover injured portions and be replaced by connective tissues within 6 weeks [68].

A collagen-based scaffold displayed repair of cardiac defect and CM regeneration when implanted into a mouse heart as a patch [69]. Temperature, pH, and shear variations can all result in transitions of polymeric phase. Collagen type I is often soluble in weak acids and produces hydrogels if neutralized [70]. Similarly, chitosan is dissolved in dilute acidic conditions, and at physiological conditions (pH = 7–7.4), it self-assembles to form a hydrogel. Drugs, growth factors, or cells are added to prepared substrates to promote regeneration [71]. Developed chitosan-aniline oligomer/dibenzaldehyde-terminated polyethylene glycol (PEG) hydrogels loaded with cells as electroactive injectable biomaterials were used for cardiac cell therapy (Figure 2) [73]. Even though biopolymers show great promise for cardiac tissue repair and regeneration, there are still challenges to solve before clinical trials in real-world treatments. Key elements, such as choosing and distributing materials, injection dose, and time, are crucial for cardiac fixing and require further investigation. And, importantly, investigation in animals that are similar to humans is essential to find how these treatments work over the long term and make sure they are both safe and effective.

3.5 Nerve tissue regeneration

The nervous system is composed of the central nervous system (CNS) and the peripheral nervous system (PNS). Nervous system regeneration is a difficult subject in tissue engineering due to the restricted potential for self-regeneration of neural tissues [74, 75]. Neural damage has an impact on life quality and can be caused by accidents, strokes, or neurodegenerative diseases, including Alzheimer’s, Huntington’s disease, multiple sclerosis, and Parkinson’s [75, 76, 77]. Astrocytes in the CNS generate glial scars as a result of complicated responses, preventing injured tissue healing. Schwann cells (SCs) are able to myelinate axons in the PNS, which aids in axon digestion (phagocytosis) during damage, resulting in neuronal regeneration. Following the lesion, SCs target neurons to form a tunnel effect that serves as a guiding way for axon repair, similar to an endoneurial tube. Studies have focused on identifying appropriate techniques to avoid further injury while stabilizing the wounded area. Sensory and motor function deficiencies in the PNS lead to paralysis of the impacted limb and intense neuropathic disorder [78, 79, 80]. The PNS lesions, when minor, can self-repair under favorable conditions. Wallerian degeneration begins after nerve injury when cellular/molecular activity spreads from distal to proximal parts of the nerve. This event results in the axoplasmic breakdown of microtubules and microfilaments, which is accompanied by changes in the nerve cell’s nucleus and increased protein synthesis to promote repair during a week [79].

SCs are essential for nerve function and undergo major alterations following damage. They transition from myelinating nerve cells to regenerative mode, breaking down the myelin coating. This process requires particular signaling molecules and cytokine release, which recruit macrophages to remove debris. Macrophages play a significant role in eliminating myelin debris and stay at the damage site for an extended duration [81, 82, 83]. SCs proliferate and align to form structures known as Büngner Bands, which serve as a scaffold for axonal regrowth. These Büngner Bands direct the regenerating axons, which extend at a pace of 1–3 mm per day, eventually leading to axonal expansion and probable functional restoration [84]. End-to-end neurorrhaphy is the significant standard for treating lesions in the PNS that are less than 1 cm in size, but autologous nerve grafting is required for bigger lesions. However, insufficient nerve transplants, neuroma formation, and immune system responses are all complicated problems with autologous grafting. As a result, the discovery of new neural therapy techniques is clinically crucial.

Polymers and conductive substrates, such as nanostructures, scaffolds, and hydrogels, are critical components in neural tissue engineering. Cell therapy, drug, gene, and growth factor delivery systems are examples of novel techniques for treating and regenerating nerve tissue lesions [85]. Derivatives or composites of chitosan, gelatin, collagen, cellulose, SF, and alginate are among the most commonly used biopolymers in nerve tissue development. Tissue engineering aids in the control of cell behavior and proliferation by creating a synthetic ECM employing biomaterials for cell culture and tissue regeneration. Hydrogel-based scaffolds can be used to create suitable three-dimensional (3D) microenvironments that mimic the extracellular environment for cell growth, promoting cell proliferation, attachment, and neuron survival [86]. The scaffolds should be biocompatible, biodegradable, safe, and anti-inflammatory, with sufficient porosity and mechanical strength to support cell growth and adhesion.

A study found that a HA-based hydrogel scaffold increased sorted embryonic stem cell-derived neural stem cell differentiation to oligodendrocytes, reduced inflammatory response, inhibited glial scar formation, and improved movement and spinal tissue integrity [87]. Another study found that combining agarose with conductive segments such as oligoaniline increased PC12 cell adhesion and proliferation [88]. Agarose promotes good cell adhesion qualities. Wen et al. discovered that a composite of nylon microfibers in an agarose matrix and subcellular filaments with sizes ranging from 5 to 30 μm can lead to the production of aligned and neurite growths in sympathetic neurons [89]. Furthermore, blends of agarose, alginate, chitosan, and aniline oligomers were employed to treat neurological disorders by developing an electrosensitive drug delivery platform. The resultant hydrogel facilitated the MSCs differentiation into dopaminergic neuron-like cells for dopamine supply in the CNS [90]. In another investigation, He et al. discovered that chitosan increases the growth and synthesis of proliferating cell nuclear antigens, as well as the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling systems in rat SCs [91].

Nerve regeneration is a complicated procedure that requires multiple components, signaling signs, and design substrates to be effective. Biomaterials have the high potential to contribute to nerve tissue healing after surgery; however, there are still problems, such as conductivity, biocompatibility, and biodegradability. Their practical application in clinical applications is constantly being investigated, and it is expected that as technology advances, these obstacles will be efficiently solved, paving the way for future progress in this sector.