Cartilaginous tissue is a type of connective tissue that creates structures such as the nasal septum or auricle, provides scaffolding for trachea and bronchi, but most of all, plays an important role in forming bone articulations [26]. The cartilage covers their connection points, taking a part in forming joints and therefore alleviating some movement-related overloads [27]. Because of not being vascularised nor innervated, cartilage has very limited regeneration potential and so the continuous friction and pressure lead, over time, to its wear and damage. SF may find its use in therapy of exactly such damages [28,29].

For regenerative therapy of cartilaginous tissue, many different forms of SF scaffolds can be used—mats, films, hydrogels, fibres, sponges or “bioinks” for 3D printing [30]. In general, all aforementioned forms of this polymer increase secretion of intercellular matrix of the cartilage as well as provide a basis for chondrocytes to grow and proliferate [29]. This last process can be additionally improved by using growth factors (GF), such as Rb1 or TGF-β1, built into the SF scaffolding [28,29]. Activity of GF, for example above-mentioned TGF-β1, can be regulated by using nanoparticles of chitosan—a polysaccharide derivative of chitin—during SF scaffold formation. This solution has been tested in vitro and in vivo in regenerating rabbits’ knee joint cartilage [31]. Things are similar when it comes to bone tissue regeneration, but more on that in the next chapter.

Creating scaffolds for rebuilding cartilaginous tissue is not the only medical use of SF. It has been shown that SF can also encourage canine adipose-derived multipotent mesenchymal stromal cells (cADMMSC) differentiation into chondrocytes. When cultivated in a standard cell culture medium, on a basic polystyrene surface, cADMMSC have the ability to differentiate into chondrocytes, osteocytes or adipocytes. However, when grown in a standard medium on a surface of SF film and scaffold, despite not using any specific chondrocyte medium, the cells strongly preferred differentiating into chondrocytes [32]. Thus, the method offers the possibility of using SF to repair cartilage injuries with patient’s own adipose-derived stem cells.

Moreover, recent studies have shown that SF scaffolds enriched with kartogenin (KGN), a small-molecule compound, reported to encourage chondrocyte differentiation from bone marrow-derived stem cells [33] and to exhibit anti-inflammatory properties [34] were able to promote cartilage tissue regeneration in rats. In a new study, SF served as a scaffolding for KGN-containing liposomes, allowed its slow release and provided mechanical support for the healing cartilage defect, while KGN enhanced extracellular matrix secretion and chondrocyte proliferation, as well as alleviated oxidative stress in the damaged area which, combined, resulted in an increased cartilage restoration score (both in micro- and macroscopic observation), compared to the control group [35]. Therefore, the usage of SF and KGN scaffolds may probably be a way to reduce negative effects of oxidative stress in cartilage injuries and disorders and make healing easier and faster for both the animal and human patients.

Another way of repairing cartilaginous tissue with SF is using a hydrogel with built-in particles of etanercept [36], an immunosuppressive agent, used in rheumatoid arthritis treatment in humans, tested on animal models [37]. To investigate this solution, a dedicated hydrogel containing SF and pullulan (an exopolisaccharyde produced by Aureobasidium pullulans yeast [38]), with addition of etanercept, has been prepared. The hydrogel was incubated in an appropriate buffer and implanted into an osteochondral defect in a rabbit knee joint, which has been made beforehand under laboratory conditions. After 8 weeks, the injuries healed completely and correctly in all test animals, which potentially gives a chance of treating cartilage defects in animals and humans in an innovative way, reducing the body’s immunological response to exogenous therapeutic compounds [36].

Similar results have been obtained using a composite scaffold of collagen and SF embedded with poly(lactic-co-glycolic acid) (PLGA) microspheres containing TGF-β1 [39]. The combination of SF with a natural polymer (collagen) and synthetic but highly biocompatible nanocarriers have encouraged bone marrow stem cells to differentiate into chondrocytes and thus stimulated cartilage repair in the rabbit knee joint [40]. This suggests that, in the future, such scaffolds could potentially help regenerate cartilage defects in human and animals, without causing unnecessary damage via more invasive methods and scarring.

It is also possible to use 3D printing methods for the regeneration of cartilage tissue. The study on developing regenerative implants inducing osteogenesis and chondrogenesis, addressing hip dysplasia, highlights the valuable attributes of SF. The innovative implant, manufactured using advanced 3D printing techniques, incorporates SF to actively stimulate chondrogenesis. Notably, the implant exhibits strong biocompatibility, as verified through various assessments. Through the integration of 3D printing and SF, this regenerative implant effectively triggers osteogenic and chondrogenic responses, ultimately restoring the natural form of the acetabulum. Consequently, it offers a promising avenue for the treatment of hip dysplasia [21].

It is worth mentioning that, apart from regenerating cartilaginous tissue with SF scaffoldings, its extracellular matrix (ECM), previously separated from the cells, can be used, combined with SF, to repair injured bones. This solution is possible because bone formation occurs mostly on a cartilaginous base (endochondral ossification) [26]. Fibroin scaffold, enriched with cartilaginous tissue ECM, although for the moment only in in vitro studies, may be used as a foundation for bone tissue regeneration [41], along with other methods described further.

In summary, SF itself is an excellent scaffold for regenerating cartilage; however, combining or embedding it with different substances might have beneficial effect on the healing tissue. For example, by adding TGF-β1 to the scaffolding chondrocyte proliferation can be increased, which is quite desirable in a sparsely vascularised tissue. Enriching it with kartogenin may support the healing process by reducing oxidative stress, which has destructive impact on cartilage tissue and is still an actual problem, and combining SF with etanercept allows it to serve its purpose without causing potential harm via inflammatory reaction. Finally, SF scaffoldings by themselves can potentially drive patients’ adipose-derived stem cells to differentiate into chondrocytes, which reduces the problem of finding a donor, and combined with collagen, embedded with PLGA/TGF-β1 microspheres, can cause bone marrow stem cells to differentiate into chondrocytes and proliferate to restore normal structure of the cartilage, without leaving any scars that would disrupt its proper functioning.

Bone tissue, like cartilage, is classified as connective tissue. It is composed of both mineral and organic substances. Bones have many key functions in the body—they store important elements, produce the bone marrow and mechanically protect internal organs. They also play an important role, together with the already mentioned cartilage tissue, in locomotion of the body [42]. Silk, extracted from the cocoons of Bombyx mori [8], has also become the focus of research into the treatment of bone damage. Developing the available knowledge on such therapies is extremely valuable, as bone damage is among some of the most common injuries with which a patient is referred to the operating theatre. The prevalence of this type of pathology, and the need to expand the available literature in this area, is evidenced by the sheer development that animal orthopaedics has experienced in recent years.

The desire to recreate the structure of bone requires consideration of its composition and the role it plays in the body. Investigating the use of silk in the treatment of bone tissue damage was therefore focused on how to balance the bioactivity with the mechanical properties of the printed bones [43]. A number of compounds, such as tricalcium α-phosphate (α-TCP) or hydroxyapatite (HA) [44,45,46], have been used to make bioplastics for cell scaffolds, but, interestingly, it has been shown over time that adding silk to these compounds improved the properties of the final product [43]. The structure of some biomaterials allows the generation of special 3D fibrous structures, which are supposed to mimic the natural ECM. The generation of such structures is made possible, for example, by the electrospinning method, which allows fibres to be produced in different spatial orientations and enables cell culture proliferation to be controlled [44]. It is a versatile technique that enables the production of nanofibre-based biomaterial scaffolds, which then mimic the nanoscale properties of certain ECM components in tissues [47]. In short, then, it is a method of producing micro-scaffolds, enabling control of the mechanical properties of the resulting fibres that build such scaffolds [44]. Silk is a polymer that is perfectly suitable for the production of such structures [48]. Regarding bone tissue, it is also possible to add bioactive factors and stem cells to the silk matrix to create an osteogenesis-friendly environment, which can additionally be controlled in various ways to direct cell regeneration [49], similar to what was discussed for cartilage. There are studies available confirming that the combination of SF, polyhydroxybutyrate-co-(3-hydroxyvalerate) (PHBV) and HA can provide a favourable environment for cell proliferation during bone regeneration. The role of PHBV and silk was to create conditions for cell adhesion, proliferation and differentiation, while HA was responsible for the induction of ECM production. Subsequent analyses (scanning electron microscopy combined with energy dispersive X-ray analysis (SEM/EDX), Fourier transform infrared spectroscopy (FT-IR), mechanical uniaxial tensile and compression tests, biodegradation tests and in vitro bioactivity tests) showed a smooth and homogeneous fibre arrangement and gradual biodegradation of the components used. Despite the disappearance of the fibrous components of the microenvironment, the fibrous structure of the sample itself was preserved. At the same time, preliminary in vitro bioassessment showed that after 1 and 3 days of culture, the cells adhered to the fibres, preserving their morphology while presenting a flattened appearance and elongated shape on the fibre surface [50].

In the new research, SF and HA were also combined with titanium oxide (TiO₂) to produce a bone-mimicking scaffold to support bone tissue formation. The scaffolds were cultured in vitro with MC3T3, i.e., osteoblast precursors obtained from the calvaria of mice mus musculus. The results demonstrated that the scaffolds with HA and TiO₂ exhibited very satisfying biological performance: cell adhesion, viability, proliferation, alkaline phosphatase activity and calcium content better than the scaffolds without HA and TiO₂. Moreover, they supported the synthesis of calcium secreted by cells [51].

Another, slightly earlier study also showed that a 3D porous composite scaffold obtained from biodegradable SF scaffold doped with mineralised collagen (MC) could induce bone regeneration in cranial defects in rats. The results showed that scaffolds could promote the regeneration of both new bone and the vascular system. This combination exhibited good mechanical properties and the ability to regulate the degradation rate of the scaffold. All of this provided favourable conditions for the proliferation of bone marrow mesenchymal stem cells (BMSC) and preosteoblasts (MC3T3-E1). An additional advantage is the relatively inexpensive cost of producing such scaffolds [52].

A three-component bioplastic consisting of polylactic acid, hydroxyapatite and silk was also used to produce bone clips, which had good biocompatibility and satisfactory mechanical properties [53].

Another method is the possibility of low-temperature print using a mixture composed of collagen, decellularised extracellular material and SF. The product of such printing also creates good conditions for cell proliferation and differentiation [43].

The study, which employs 3D printing technology to fabricate inventive scaffolds composed of SF, collagen and HA (SCH) infused with recombinant human erythropoietin (rh-EPO), reveals that these composite scaffolds exhibit gradual degradation, foster the proliferation of osteoblasts, and notably improve the reconstruction of bone defects, offering substantial potential for clinical applications. The use of a low-temperature 3D printing method effectively maintains the biological activity of SF and collagen, resulting in scaffolds with optimal properties [20].

Another form in which silk can be used to be effective in the treatment of bone injuries are hydrogels. Their microstructure closely resembles the structure of the tissue, which is a major advantage. For bone defects treated with hydrogels containing silk, therapeutic effects were achieved in a short time. In addition, the healing process was accompanied by other positive elements, such as increasing the thickness of the bone trabeculae or increasing the rate of mineral deposition. Nanohydroxyapatite was also added to the hydrogel, which further improved cell metabolic activity and naturally resulted in more efficient bone regeneration. Due to their biocompatibility and low immunogenicity, injectable silk hydrogels, on the other hand, can provide an ideal carrier for therapeutic agents [49]. For example, a hydrogel has been developed that, when administered, simultaneously released bioactive magnesium, silicon and strontium ions, creating a microenvironment suitable for osteo- and angiogenesis [54]. In addition, studies have also been conducted showing that the SF hydrogel can be combined with GFs, such as VEGF or BMP-2. Administration of GFs alone may result in a number of undesirable effects, such as the formation of ectopic bones. Such administration of isolated GF may also result in therapy failure, due to the short half-life of these proteins [55]. So, in practice, for the effective carriage of BMP-2, SF hydrogel was fabricated using the electrospun poly(ε-caprolactone) (PCL) nanofibre mesh tubing. The results demonstrated that this hydrogel was very effective carrier for BMP-2, and this combination resulted in promising level of bone formation. Moreover, the silk hydrogel has been completely degraded, and the repair of large bone defects in that case was satisfying [56].

SF hydrogels were also used in a study on lacunar bone regeneration. For this purpose, SF was combined with mesoporous bioglass and sodium alginate. By activation of the mitogen-activated protein kinase (MAPK) signalling pathway, the hydrogel caused an osteogenesis-friendly environment [57].

It is also possible to prepare a special silk nanofibre film using electrospinning, which is then able to mimic the microstructure of the natural ECM. A study conducted on a rabbit calvaria defect model has demonstrated new bone formation in vivo, and moreover it was found that cells on this type of substrate presented higher alkaline phosphatase activity and more efficient osteocalcin production [49]. In the production of this type of film, it is also possible to mix various polymers with SF to improve the stability and mechanical properties of the final product. A promising blend is the combination of silk with chitosan, as the properties of both polymers enable the regulation of cytokine and GF secretion in the defect and the stimulation of cell proliferation and differentiation. Interestingly, this type of combination can be used not only as an implant shell to repair bone damage, but also as a scaffold in tissue engineering to treat defects of the skin, cornea, adipose tissue or other soft tissues. Another study [58] compared the proliferation and differentiation capacity of rat bone marrow-derived mesenchymal stem cells into bone and adipose tissue cells on silk-chitosan film and on polystyrene tissue culture plates, and the results have proved that a thin film of SF and chitosan not only provided a comparable environment for growth and proliferation, but also promoted the differentiation of stem cells into bone and adipose tissue cells.

Chitosan has also been combined with SF in other, more recent studies. Two-layer multifunctional nanofibre mats were successfully produced in some research—the osteogenic side of which consists of SF/PCL and the antibacterial side of PCL/chitosan). The results demonstrated that such mats were characterised by very favourable biocompatible properties, sufficient hydrophilicity and appropriate mechanical properties, as well as high tear strength [59]. The antibacterial aspect of such mats seems to be particularly beneficial here, because this property can significantly reduce postoperative bacterial infections caused by grafts obtained in that way.

Various types of wounds and skin damages are the next extraordinarily prevalent issue in both human and veterinary medicine. The therapy of such afflictions and injuries is often a challenge, significantly impacting the quality of life for patients. This exerts pressure upon scientists and doctors to combat this issue with utmost efficacy [30]. Both acute and chronic conditions are challenging not only for physicians, but also for patients and animal owners, primarily due to the difficulty in providing an appropriate wound healing conducive environment. All forms of skin damage represent a potential threat, as this organ serves as the primary barrier between the external environment and the organism. Disruption of this barrier is an open gate for pathogens, which can be a lethal threat [10,60]. In animals, it is particularly challenging to maintain dressings, change them (if the animal is hyperactive, aggressive or wild) and prevent the patient from accessing the site of injury. It is therefore extremely important that the healing process proceeds quickly, without discomfort or complications and with the least possible interference from the veterinarian or owner. In addition, in both animals and humans, it is common to encounter wounds that are extensive, deep or difficult to heal due to a variety of factors, such as associated diseases, e.g., diabetes, or immunological deficiencies [9,12,13,24,61]. Such damages necessitate specialised treatment and materials that adequately safeguard the injury environment and provide a matrix for tissue regeneration, thereby facilitating wound closure [30].

To produce economically viable and thus widely accessible medical materials based on silk for wound treatment, two types of three-dimensional fibroin structures are employed: nanofibrous mats and micro-porous scaffolds. These structures are subsequently coated or combined with specific agents aimed at conferring bioactive characteristics [9]. Such agents can include other proteins secreted by silkworms, like sericin, antibiotics, anti-inflammatory factors, or agents promoting cell proliferation and migration [13,25,30].

All chemical, biological, or genetic modifications aim to achieve precisely defined, highly functional traits that underlie the therapeutic properties of silk-based materials. These modifications result in various types of dressings and fillers that create an environment around injuries and within wounds, facilitating and expediting the entire wound and skin damage healing process. Therapeutic materials used in treatment must exhibit low immunogenicity. SF has this characteristic, as well as many others that account for its therapeutic value. Through functionalisation using additional factors, scaffolds, mats, aerosols, fillers, films, or hydrogels produced from silk proteins, further desired medical attributes are instilled. These attributes include biocompatibility, biodegradability, versatility, and stability in both intra- and extracellular environments.

Genetic engineering is one of the methods of obtaining SF with specific characteristics. The process of gene recombination significantly facilitates and accelerates the production of functional 3D matrices from fibroin. Thanks to genetic engineering, the production of SF with the desired characteristics is much faster and more efficient. The most efficient method of producing functional fibres is based on the synthetic coding of the relevant amino acid sequences. Recombinations leading to silk fibre functionalisation can be achieved not only through alterations in the fibroin amino acid sequence, but also by adding functional proteins [23]. Appropriate variations allow control of potential therapeutic agents of a future matrix composed of recombinant silk proteins [13]. Bioengineering also enables the incorporation of non-therapeutic (but equally important) features, such as faster degradability or the ability to change conformation or a state of aggregation under specific stimuli [14], which has already been mentioned in the fragment about bone and cartilage therapy. These modifications contribute to enhanced biocompatibility, primarily due to an increased capacity for cell attraction and adhesion to the matrix [12,13,60]. Studies conducted on mice and rats have demonstrated that nanofibrous structures enriched with molecules that promote cell adhesion, such as integrins, not only facilitate cell adhesion but also promote cell proliferation, accelerate neovascularisation, and enhance tissue healing [13,60,62,63]. Another pivotal aspect of silk fibre functionalisation involves conferring antibacterial properties. Antibacterial factors hold immense importance in the preparation of wound dressings, graft matrices, or silk-based fillings, as they prevent infection development, thus providing a suitable environment for healing and significantly speeding up the recovery process [13,23,64]. The fibres forming therapeutic 3D structures can be enriched with antibiotics, as well as proteins like defensins or hepcidins [23]. Coating fibroin with antibacterial agents is particularly crucial because non-functionalised silk material in this regard could foster microbial colonisation within it, thereby precluding its therapeutic properties [13]. Another additive imparting desired traits to fibroin can be other molecules, both organic and inorganic, such as silaffin (which facilitates the deposition of silica on the biomaterial surface, promoting cell proliferation matrix formation and stabilisation) [65], gelatine or glycol, calcium ions, sodium chloride, and glucose (known as porogens, which support the formation of pores in nanofibrous scaffolds) [25,30,65].

There are numerous types of dressings produced by enriching silk nano- and microfibres, including mats, scaffolds, films, hydrogels, spheres, matrices, coverings, aerosols, and sponges [23,25]. They have a very wide range of applications, from uncomplicated traumatic wounds, post-operative wounds, deep loss of large skin areas, diabetic wounds, and burns, to advanced and extensive skin grafts. Comparative studies between functionalised silk dressings and other wound dressings have demonstrated that enriched fibroin reduces the expression of pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines in burn cases [13]. Another study showed the positive impact of fibroin sponge dressings on the healing of challenging diabetic wounds [61]. The creation of effective hydrogels using SF represents a significant advancement in the field of wound repair. Renowned for its robust biocompatibility, SF serves as a fundamental building block for constructing these hydrogels. One of its key attributes is controlled biodegradability, allowing for precise customisation to meet specific tissue regeneration needs, such as promotion of vascular regeneration, which regulates the local immune response. These hydrogels benefit from SF’s excellent mechanical properties, enhancing their stability and overall efficacy. SF’s exceptional self-assembly capabilities play a pivotal role in developing hydrogels with remarkable stability, making them essential components in tissue engineering and regenerative medicine. This innovative approach opens new avenues for designing biocompatible, customizable hydrogels that hold great promise for various applications in the field of healthcare and biomedical research [66,67].

Additionally, some innovative techniques in tissue engineering and regenerative medicine such as the 3D printing highlight the ongoing efforts to enhance wound healing treatments. By incorporating bioengineered materials like SF and gelatine hydrogel into the development of artificial skin, researchers are actively working to create more effective wound healing solutions for both human and veterinary medicine. These advancements in bioengineered hydrogels and 3D printing techniques contribute to the diverse range of therapeutic materials available for wound care, ultimately accelerating the healing process and improving patient outcomes [19,22].

Silk has been used for centuries in the skin damage treatment, and today, there is an increasing focus on studying its therapeutic properties and actively seeking new technologies and applications. Research efforts are expanding not only in animal models, but also in human trials, and their results demonstrate the positive impact of utilising SF in treatments. These findings underscore silk’s potential as a promising material not only in medicine, but also in other fields such as the food and cosmetic industries [65]. In summary, the goal in wound healing for both human and veterinary medicine is to effectively accelerate the healing process, thereby enhancing patient comfort and treatment efficacy. Silk proteins serve as excellent material, and when functionalised, they acquire numerous characteristics conducive to healing even the most challenging skin damage. Various types of dressings are produced by enriching SF fibres with diverse factors that positively influence the recovery process. Additionally, its widespread availability and relatively low production costs are crucial features. This accessibility ensures that new technologies and therapeutic material production are feasible worldwide, bridging the gap between developed and developing countries.