Collagen in Orthopedics: From Molecules to Therapies

Abstract

Collagen, the primary constituent of the extracellular matrix (ECM) in most living organisms, is a structurally unique protein that has been classified into seven categories based on its supramolecular structure. The abundance of collagen in the human musculoskeletal system implicates it in the pathogenesis of several orthopedic conditions. Consequently, its metabolic products are useful biomarkers for the prognosis, diagnosis, and monitoring of orthopedic ailments. Collagen also finds therapeutic applications in orthopedics because of its biocompatibility, biodegradability, and mechanical stability. Several collagen-based biomaterials (CBBs) including sponges and nanofibers are currently used in orthopedic therapy. This chapter begins with a concise description of the biosynthesis of collagen as well as its classification and distribution in the human body. Subsequently, the chapter discusses the potential of collagen in orthopedic diagnostics and therapeutics while also delineating the challenges posed by collagen-based biomarkers, the risks associated with collagen from different sources, and the drawbacks of the conventional methods used to fabricate CBBs. Finally, the chapter explores the use of modern techniques like 3D bioprinting for the synthesis of highly structured collagen matrices and emphasizes the need for future research into collagen-based diagnostics and therapeutics in orthopedic surgery.

1. Introduction

Collagen is a morphologically diverse biopolymer found extensively in the extracellular matrix (ECM), a structural network found in most eukaryotic cells that consists of non-fibrillar amorphous substances like glycoproteins and fibronectin as well as fibrillary components such as elastin and reticular fibers [1, 2]. Collagen is the most abundant human protein and is largely present in the ECM of multiple tissues including the skin, cartilage, bones, and blood vessels. Up to 35% of the total protein mass in mammals corresponds to collagen [3, 4]. Based on their structure, function, and composition, 29 different types of collagens have been elucidated, each designated by a Roman numeral. Type I collagen is the most common type found in most tissues, especially skin, bones, and tendons, followed by type II collagen, which is restricted to the cornea and the hyaline cartilage in joints, and type III collagen, which is mainly seen in blood vessels [5]. As diagrammatically represented in Figure 1, collagens can be classified into seven categories based on their supramolecular assembly, namely, fibril-forming collagens, fibril-associated collagens with interrupted triple helices (FACITs), network-forming collagen, transmembrane collagens, endostatin-producing collagens, anchoring fibrils, and beaded-filament forming collagens [6].

Figure 1.

Classification of collagens based on supramolecular assembly [6].

As shown in Figure 2, the biosynthesis of collagen begins in the nucleus with the transcription of specific COL genes encoding the pro-α1 and pro-α2 polypeptide chains. These genes are particular to each collagen type—for instance, COL1A1 and COL1A2 encode type I collagen, while COL2A1 encodes type II collagen [8]. The transcribed mRNA moves into the cytosol where the ribosomes translate it to produce pre-procollagen chains, which are then translocated into the lumen of the rough endoplasmic reticulum, where they are converted into procollagen chains by undergoing three major post-translational modifications—removal of the N-terminal signal peptide, hydroxylation of lysine and proline by hydroxylase with vitamin C as the co-factor, and glycosylation of lysine hydroxyl groups with glucose and galactose. Three procollagen chains then wind around each other to form a coiled-coil triple helical structure, which moves to the Golgi apparatus, where it is packaged into secretory vesicles and transported to the extracellular space. Procollagen is converted to mature tropocollagen via cleavage of the C-terminal and N-terminal pro-peptides by collagen peptidases. Finally, in the case of type I collagen, five tropocollagen molecules assemble into a cross-linked network via covalent bonding by the action of lysyl oxidase on the lysine and hydroxylysine residues to form a supramolecule known as the collagen microfibril. These microfibrils are organized into collagen fibrils and fibers that provide structural integrity to the body tissues [6, 7]. All collagens are characterized by the presence of the Gly-X-Y motif, a repeating amino acid sequence that comprises glycine and, generally, proline and hydroxyproline as X and Y, respectively. Fibrillar collagens consist of a high percentage of uninterrupted Gly-X-Y motifs, which contributes to the stability of their fibrillar structure. The intramolecular hydrogen bonds between glycine residues impart tightness to the triple helix while proline and hydroxyproline confer resistance against proteases, thus limiting hydrolysis and maintaining structural integrity by virtue of their acyclic nature [9, 10]. Thus, they display structural, mechanical, and tissue-building properties through the regulation of critical cellular processes like proliferation, differentiation, and migration [5].

Figure 2.

Schematic representation of collagen biosynthesis [7].

The bone is primarily a rigid, connective, osseous tissue that has two main parts—an inorganic component that constitutes 65% of the bone, comprising hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂] crystals and mineral ions like sodium and potassium as well as an extracellular organic matrix known as the osteoid, 90% of which is made up of type I collagen while the non-collagenous organic molecules include proteoglycans, osteonectin, and osteocalcin. The four cell types in the osteoid are osteocytes, bone lining cells, osteoblasts, and osteoclasts. Osteoblasts are present on the bone surface and produce type I collagen. They also deposit hydroxyapatite (HA) crystals on collagen molecules to promote matrix calcification. HA provides rigidity, while collagen provides tensile strength to the bone. Type I collagen accumulates along mechanical stress lines in the osteoid to create a structural framework for mineralization [11, 12]. Errors in collagen metabolism can lead to a variety of orthopedic disorders like osteogenesis imperfecta, Ehlers-Danlos syndrome (EDS), and certain types of skeletal dysplasia including spondyloepiphyseal dysplasia congenita (SEDC) and otospondylomegaepiphyseal dysplasia (OSMED) [4]. Osteogenesis imperfecta is a genetic disorder characterized by bone embrittlement due to a mutation in type I collagen while EDS leads to heritable connective tissue disorders (HCTD) caused by mutations in specific types of collagens [13, 14]. For instance, EDS type IV is an autosomal dominant disorder caused by the mutated COL3A1 gene encoding type III procollagen [15]. Skeletal dysplasia, also known as osteochondrodysplasia, includes several genetic conditions that affect the bone and cartilage by different means. SEDC is a type II collagen disorder while OSMED is caused due to problems in type XI collagen [16].

The first reported collagen-based biomaterial (CBB) was the “catgut,” a collagen-rich suture material developed in 1881 by Joseph Lister, the father of modern surgery [17]. Given its biocompatibility, tensile strength, biodegradability, mechanical stability, and flexibility, collagen is an excellent therapeutic biomaterial. Currently, various CBBs like sponges, hydrogels, microspheres, nanofibers, and injectable solutions are commercially used for several applications including surgery, wound healing, oral therapy, tissue regeneration, and drug delivery [18]. Furthermore, collagen is a viable biomarker since it is abundantly present in most tissues and can be detected in blood plasma and tissue samples. The metabolic products of collagen are used to diagnose and monitor several conditions including cancer [19, 20].

This chapter focuses on the applications of collagen in orthopedic surgery. Orthopedics refers to the diagnosis, treatment, and prevention of injuries and disorders affecting different components of the musculoskeletal system. Being a predominant protein in the bone and cartilage, collagen and its metabolic products are widely used as diagnostic biomarkers for the detection of orthopedic conditions that require more efficient monitoring methods including ankylosing spondylitis, osteoarthritis, and rheumatoid arthritis. The orthopedic applications of collagen-based diagnostics have been delineated in the following section of the chapter. The next section discusses the use of collagen in orthopedic therapeutics such as sponges, hydrogels, and microspheres for the treatment of bone injuries, cartilage defects, athletic strain, and disorders like rheumatoid arthritis. The subsequent section concentrates on the challenges associated with the use of collagen in orthopedics including the drawbacks of different sources of collagen and the shortcomings of the techniques employed for its extraction, cross-linking, and sterilization. It also explores modern technologies such as plastic compression, electrospinning, and 3D bioprinting that have been introduced in recent years for fabricating highly structured CBBs. The chapter emphasizes the need for further research into the clinical applications of collagen-based diagnostics and therapeutics in the field of orthopedics.

2. Collagen-based orthopedic diagnostics

Due to its extensive presence throughout the ECM of the musculoskeletal system, the collagen network is affected during the pathogenesis of several orthopedic conditions. Thus, the levels of collagen degradation products serve as diagnostic indications of certain orthopedic disorders, especially those affecting the articular cartilage such as osteoarthritis (OA) and rheumatoid arthritis (RA). The hyaline articular cartilage is a connective tissue lacking nerves, blood vessels, and lymphatics that is present between joints. It is made up of chondrocytes and has a dense collagenous network in the ECM that is rich in collagen type II followed by types IX and XI, while types III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII are present in minor quantities. Based on collagen arrangement and chondrocyte proximity, the articular matrix is divided into three zones—the pericellular matrix (PCM) surrounding the chondrocytes, the territorial matrix surrounding the PCM, and the interterritorial matrix, which forms the largest zone. Type I and type III collagens are predominant in the peri-articular soft tissues, while the PCM consists of type III–VI collagens [7, 21]. During disease conditions, the articular cartilage gets damaged, leading to collagen degradation in the ECM by collagenases like A disintegrins and metalloproteinases with thrombospondin motifs (ADAMTS), cathepsins, and matrix metalloproteinases (MMPs), mediated by inflammatory molecules. Several fragments are released into the systemic circulation and the synovial fluid due to collagen degradation. Thus, these fragments act as highly specific and sensitive biomarkers indicative of the underlying clinical conditions. Table 1 encapsulates the distribution of collagens in the articular cartilage and the proteinases that degrade them, especially during orthopedic conditions [22, 31].

Collagen type	Distribution in articular cartilage	Collagen-degrading proteinases	References
Type I	Fibrocartilage, elastic cartilage	MMP-2	[23]
Type II	ECM of all zones	MMP-1, 3, 13	[24]
Type III	PCM	MMP-3	[25]
Type IV	PCM	MMP-2, 9	[23]
Type V	PCM	MMP-2, 9	[26]
Type VI	PCM	MMP-2, 9	[27]
Type IX	Growth-plate cartilage	MMP-3, 13	[28]
Type X	Calcified zone and hypertrophic cartilage	MMP-1, 3, 13	[29]
Type XI	Articular cartilage	MMP-2	[30]
Type XII	Cartilage with more organized fibril orientation	Not available
Type XIV	Uniformly throughout the articular cartilage	MMP-13	[22]
Type XVI	Territorial matrix of chondrocytes	Not available
Type XXII	Articular surface of joint cartilage
Type XXVII	Proliferative zone chondrocytes

Table 1.

Distribution and susceptibility of collagens in the articular cartilage [22].

2.1 Ankylosing spondylitis (AS)

Ankylosing spondylitis (AS), also known as axial spondyloarthritis (axSpA), is a systemic, chronic, inflammatory autoimmune condition brought about by genetic and environmental factors that affects the spine, sacroiliac joints (SIJs), and adjacent soft tissues. It is characterized by joint inflammation, fibrosis, calcification, and ultimately, a progressive increase in spinal rigidity resulting in loss of flexibility and movement due to complete fusion of the spine [32]. While the pathophysiology of AS is yet to be elucidated, it is associated with the HLA B27 gene polymorphism [33]. The onset of AS begins with immune dysregulation leading to chronic inflammation, brought about by the interleukin-17/23 (IL-17/23) pathway and tumor necrosis factor (TNF) as well as other pro-inflammatory cytokines such as IL-6, IL-10, IL-22, and interferon γ (IFNγ). This is followed by matrix erosion caused by MMP-1 and cathepsin K. Finally, bone fusion occurs during syndesmophyte formation by the action of the bone morphogenetic protein (BMP) and Wnt signaling pathway, the master regulator of bone remodeling [34]. Currently, C-reactive protein (CRP) is the only inflammatory marker that is clinically used to diagnose axSpA. There is an exigent need for sensitive biomarkers that facilitate effective diagnosis and monitoring of AS [35]. During AS, the cartilage intermediate layer protein 1 (CILP-1) produced by chondrocytes for the reduction of ECM proliferation is cleaved by MMP-1, MMP-8, and MMP-12 leading to increased levels of CILP-M, an MMP-generated neoepitope of CILP-1 [36]. Collagen fibril formation is disrupted in AS by the action of MMP-2 and MMP-9 which degrade type V collagen to produce C5M fragments. Thus, elevation of C5M levels also serves as an indicator of AS [26]. Further, due to extensive ECM remodeling, MMP-mediated fragments of collagen types I, III, IV, and VI, namely, C1M, C3M, C4M2, and C6M, respectively, are present in higher amounts in AS patients, thereby facilitating their use as blood-based biomarkers for tracking disease severity and progression [37].

2.2 Osteoarthritis (OA)

Osteoarthritis (OA) is a chronic, degenerative joint disease associated with aging. It is characterized by cartilage degradation, bone remodeling via BMPs, synovial membrane inflammation, subchondral osteosclerosis, synovial angiogenesis, and osteophyte formation by the action of vascular endothelial growth factors (VEGFs). As OA progresses, chondrocytes display a senescence-associated secretory phenotype (SASP) wherein they display mitochondrial dysfunction, leading to oxidative stress and ultimately undergo senescence. The cartilage breakdown products are phagocytosed by synovial cells which release pro-inflammatory and catabolic molecules including cytokines like TNFα and IL-6, chemokines, neuropeptides, bioactive lipids like prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), as well as adipokines like lipocalin, leptin, and adiponectin [22, 38, 39]. Though chondrocytes produce anti-inflammatory mediators like IL-3, IL-4, and IL-1Ra as well as tissue inhibitors of metalloproteinases (TIMPs), they are outnumbered by pro-inflammatory molecules, thus allowing synovial inflammation to increase [40]. CILP is overproduced in early OA and inhibits tumor growth factor β (TGFβ), thereby hindering ECM proliferation [41]. The pathophysiology of OA has been diagrammatically elucidated in Figure 3. Oxidative stress and low-level inflammation lead to the breakdown of the collagen network in the ECM primarily via the degradation of type II collagen by MMP-13 (collagenase-3) along with MMP-1 (collagenase-1) and MMP-3, all three of which are upregulated by lipocalin and adiponectin while leptin upregulates MMP-1 and MMP-3 production [42]. Furthermore, collagen type II production is inhibited by IL-1β, due to which reduction in its levels is suggestive of OA. The MMP-mediated cleavage of type II collagen also results in the accumulation of COL2-3/4C (short) neoepitopes and urinary C-terminal telopeptide (uCTX-II), which act as highly informative biomarkers as their levels increase with disease progression [43, 44]. Due to the increased deposition of type III collagen on the joints, the COL3/ADAMTS neoepitope, an ADAMTS-generated type III collagen fragment, serves as a serum biomarker for early OA [45, 46]. On the other hand, the presence of MMP-mediated type 3 collagen metabolites (C3M) indicates the degradation of soft tissues. Lipocalin and adiponectin increase the levels of inducible nitric oxide synthase, leading to the production of nitric oxide (NO), which causes protein nitration. Thus, increased serum concentrations of Coll 2-1 and Coll 2-1 NO₂ peptides are diagnostic OA markers [47, 48]. Leptin and lipocalin stimulate type X collagen (Col10) production, which contributes to the hypertrophy of chondrocytes, a characteristic of OA [49]. Thus, in addition to IL-1β, osteocalcin, osteopontin, and VEGF, type X collagen is also a major biomarker for chondrocyte hypertrophy [50]. Moreover, the blood serum levels of Col10neo, a cathepsin K-generated neo-epitope of Col10, signify hypertrophic chondrocytes and can be used to detect OA in the knee joints [51]. The presence of type I collagen in the articular cartilage denotes the potential onset of OA since it is a component of the fibrocartilage repair tissue that fills cartilage defects [52].

Figure 3.

Pathophysiology of osteoarthritis [40].

2.3 Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a systemic, chronic autoimmune disorder characterized by synovial joint damage, synovial inflammation, cartilage destruction, and matrix degradation [53]. As shown in Figure 4, there is an imbalance between the pro-inflammatory and anti-inflammatory cytokines in RA due to the overproduction of TNFα, IL-1β, and IL-6 by synovial tissue macrophages (STMs), leading to chronic inflammation. This stimulates the release of matrix-degrading enzymes by the chondrocytes, leading to ECM degradation. Another major characteristic of RA is the tumor-like transformation of activated synovial cells into fibroblast-like synoviocytes (FLS), highly proliferative cells that produce MMP-1, MMP-3, MMP-13, AMADTS4, ADAMTS5, and cathepsins. These collagenases cause tissue damage and promote the infiltration of immune cells like neutrophils and macrophages, which also release MMPs, ADAMTS, and pro-inflammatory cytokines, thereby exacerbating the inflammatory condition [55]. RA is characterized by several autoantigens including guanosine proteins, type II collagen, and circulating serum proteins. Autoantibodies like the rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) that are produced to counter these antigens serve as diagnostic markers of RA [22, 54, 56]. During disease progression, MMP-13 cleaves type II collagen, thus reducing its levels and leaving behind degradation products in the articular cartilage. The breakdown of soft tissues involves the MMP-mediated cleavage of type I and type III collagens, leading to the release of C1M and C3M, respectively—thus, their serum levels are used to track RA progression [57]. The metabolism of type IV collagen during destructive synovitis growth due to the tumor phenotype of FLS leads to elevated serum levels of C4M, another marker for disease prognosis [58].

Figure 4.

Pathophysiology of rheumatoid arthritis [54].

3. Collagen-based orthopedic therapeutics

As a major structural protein in the human body, collagen plays a vital role in the repair and regeneration of tissues. Therefore, collagen-based therapies have been widely studied for clinical applications in bone and soft tissue regeneration. The role of collagen in maintaining joint strength and bone health, in combination with its breakdown during orthopedic conditions, makes collagen-based biomaterials (CBBs) a viable choice for managing musculoskeletal injuries and diseases, especially those involving cartilage damage, which cannot be reversed. Generally, naturally occurring type I and type II collagen proteins are extracted from human, bovine, murine, equine, and porcine sources such as bones, tendons, and skin as well as marine sources like fish skin, scales, and bones, via ECM decellularization. The undenatured proteins are then subjected to protein hydrolysis to obtain hydrolyzed soluble bioactive peptides that have better digestibility, absorption, and distribution during oral administration. Collagen can also be produced synthetically as in the case of recombinant human collagen (rhCOL) obtained by transfecting COL genes into different prokaryotic and eukaryotic expression systems such as Saccharomyces cerevisiae, Pichia pastoris, Zea mays, and Trichoplusia ni, as well as transgenic animal models like mouse embryos and Chinese hamsters [59]. The composition, properties, and functionality of CBBs vary depending on the source of collagen and the hydrolysis process. While developing CBBs, the effect of the collagen source and extraction technique on properties such as biodegradability, immunogenicity, biocompatibility, and mechanical stability must be considered [60, 61]. CBBs are available in several forms including scaffolds, oral supplements, microspheres, and nanospheres, each of which lend themselves to specific therapeutic applications.

3.1 Collagen-based scaffolds

Collagen-based scaffolds are three-dimensional biomimetic matrices having porous, interconnected structures that facilitate the regeneration of damaged bone tissues. Two types of collagen scaffolds widely used in orthopedic therapy are sponges and hydrogels. Since the use of pure collagen does not facilitate control over scaffold properties like porosity and stability, most scaffolds use composites of collagen mixed with natural polymers like chitosan, silk fibroin, and hyaluronic acid, synthetic polymers such as polyethylene glycol (PEG), polyglycolide (PGA), and polyvinyl alcohol (PVA), as well as ceramics including hydroxyapatite, β-tricalcium phosphate (β-TCP), and silicate [62, 63]. Collagen sponges are generally developed by the process of freeze-drying, also known as ice-segregation-induced self-assembly or lyophilization. In this process, the collagen-based solution is frozen to entrap collagen molecules within hexagonal ice crystals, followed by sublimation of the water molecules, leading to the formation of intermolecular cross-links between the collagen aggregates, thus creating a highly porous, sponge-like structure, as shown in Figure 5. The shape and pore size of the sponge can be altered by controlling the fabrication parameters including duration, freezing temperature, and mold morphology. The optimal pore size must allow cell migration and nutrient diffusion while also facilitating cell attachment [64, 65]. Collagen sponges are generally loaded with growth factors like BMPs and fibroblast growth factors (FGFs) for use in bone tissue regeneration and cartilage defect repair to promote fibrin network formation in the ECM, chondrogenic differentiation of mesenchymal stem cells (MSCs), as well as osteoblast proliferation and migration [66, 67]. An atelocollagen sponge loaded with recombinant human BMP-2 (rhBMP-2) and implanted into mice models was shown to promote bone formation via osteoconduction [68]. Highly porous collagen implants were successful in treating injured rotator cuffs via connective tissue repair in human subjects [69]. Collagen sponges are also used to deliver antibiotics and biomolecules in orthopedic conditions like acute septic arthritis [70]. They are preferred due to their ease of processing, sterilization, loading, and preservation as well as their optimal functionality in inducing tissue regrowth [64].

Figure 5.

Freeze-drying process of collagen sponge formation [64].

Collagen hydrogels are hydrophilic, versatile scaffolds that can adopt a range of structures and physical properties based on factors like collagen concentration, pH, temperature, and ionic strength. Injectable hydrogels and hydrogel implants synthesized using collagen in combination with substances like hydroxyapatite, chitosan, and alginate are used for in situ bone tissue repair as they display physiological bone-like surface properties, minimal invasion, and high biocompatibility [71]. Given their highly absorbent, water-rich micro-environment and suitability for cell encapsulation, they provide an efficient biomimetic substrate for carrying adult human bone marrow-derived stem cells (hBMSCs), thus promoting ECM development and bone regeneration. They are also viable carriers of growth factors like rhBMP-2 and therapeutic drugs like doxycycline in orthopedic conditions since they form a continuous, direct delivery system and ultimately promote osteogenesis [72, 73, 74].

3.2 Collagen-based oral supplements

Oral supplementation is a popular form of collagen-based therapy, given its biocompatibility, digestibility, and bio-accessibility. Upon oral administration in the hydrolyzed form, collagen gets digested in the gastrointestinal tract and releases amino acids and peptides in the small intestine, which then enter the bloodstream. The high percentage of proline and hydroxyproline in collagen protects the peptides from intestinal proteolytic enzymes, thereby preserving their bioactivity. The two forms of collagen in oral supplements are undenatured type II collagen (UC-II), which retains its biological activity, and partially hydrolyzed collagen, which is degraded enzymatically or physically using heat/pH. Hydrolyzed collagen extracted from eggshell membranes reduces the levels of CTX-II and other inflammatory biomarkers [75]. Oral supplementation of collagen has been widely used to treat the symptoms of OA and RA including inflammation and joint pain. While both forms of collagen are effective, the analgesic benefits conferred by hydrolyzed collagen supplements were seen to be stronger in the case of OA, potentially due to better absorption [76]. The administration of an oral preparation comprising hydrolyzed collagen type II in combination with bioactive oligopeptides of natural hydrolyzed keratin, chondroitin sulfate, and hyaluronic acid was shown to reduce synovial fluid levels of the inflammatory cytokines, IL-6, IL-8, IL-10, and alleviate joint pain in patients with knee OA, thereby demonstrating its viability as an adjuvant orthopedic therapy [77]. Daily oral consumption of hydrolyzed type 1 collagen (hCol1) is another potential strategy to reduce pain and inflammation via chondrocyte protection during post-traumatic OA [78]. Collagen peptides are also viable analgesics in orthopedic treatments—they significantly reduce knee pain in OA patients through the chondroprotective effect brought about by reducing the expression of MMP-13, thereby inhibiting the degradation of type II collagen [79, 80]. The oral form of chicken type II collagen is effective in inducing oral tolerance and subsequently reducing inflammation, pain, joint swelling, and stiffness in RA patients [81, 82]. Type II collagen-mediated oral tolerance is also a viable treatment for collagen-induced arthritis (CIA) as it causes an anti-inflammatory effect by increasing the production of anti-inflammatory IL-10, ultimately decreasing joint swelling and pain levels [83]. Bovine type II collagen induces oral tolerance in CIA, leading to reduced levels of pro-inflammatory IL-17 and subsequent downregulation of the IL-17 stimulated expression of receptor activator of nuclear factor κB ligand (RANKL), an osteoclastogenic mediator, in CD4⁺ T-cells [82]. Bioactive collagen peptides (BCP) are also used as oral supplements for alleviating knee pain [84].

3.3 Collagen-based microspheres and nanospheres

Collagen-based microspheres are spherical microstructures ranging in diameter from 3 to 40 μm. They are produced by the emulsification of native collagen in organic solvents like paraffin followed by glutaraldehyde-based cross-linking [85]. Mesenchymal stem cells encapsulated in collagen microspheres are used in articular cartilage repair since they promote chondrogenic differentiation and hyaline cartilage-specific gene expression. Further, the condensed collagen meshwork in microspheres is more stable with higher cell density as compared to collagen gels, and its superior chondroconductive capacity facilitates remodeling of the type I collagen template into a biomimetic, ECM-like matrix comprising type II collagen, glycosaminoglycan (GAG), and aggrecan [86]. Collagen microspheres can also efficiently maintain a cellular network of human osteoarthritic chondrocytes (hOACs). As shown in Figure 6, hOACs were enzymatically isolated from cartilage tissue samples of OA patients and encapsulated into collagen microspheres. The 3D hOAC-collagen microsphere model was shown to recapitulate the OA phenotypes more efficiently than the traditional 2D monolayer cultures and 3D pellet cultures in terms of physiologically relevant cell density, ECM interactions, MMP-13 levels, and extracellular deposition of type II and type X collagen. Thus, collagen microspheres are useful three-dimensional in vitro models for studying OA and screening potential therapies [87]. Fibrillar collagen microspheres of different sizes and morphologies can be prepared using water-in-oil emulsions by controlling the hydrophilic-lipophilic balance (HLB) and promoting the fibrillogenesis and gelling of collagen molecules, thus negating the need for cross-linking. These injectable microstructures are highly stable and biocompatible, and they mimic the ECM micro-environment, which is why they find application in the treatment of bone injuries and cartilage defects, especially in drug delivery systems [88]. Collagen microspheres are widely used in bone tissue regeneration as carriers of antibacterial drugs, glucosteroids, and growth factors like BMP and VEGF since their degradation rate can be controlled via chemical modifications like cross-linking thus facilitating the time-controlled, systematic release of the encapsulated molecules [89].

Figure 6.

Synthesis and evaluation of a 3D hOAC-collagen microsphere model [87].

Collagen nanospheres are spherical structures with a diameter in the nano-range. They are generally synthesized using a high-voltage electrostatic field via self-assembly and fibrillogenesis. They can be used as drug delivery systems and as carriers of growth factors and other bioactive molecules. They also form a major component of scaffolds and implants used in bone tissue regeneration since they can effectively encapsulate stem cells and promote cell differentiation and proliferation [89, 90]. Factors like temperature and collagen type impact the size and structure of the nanospheres. Temperatures below 37°C are generally favored, and type II collagen is preferred for maintaining the spherical structure of microspheres. While type I collagen yields nanospheres of relatively smaller sizes, the sphericity of type II collagen nanospheres is better, due to which the latter is generally preferred for use in CBBs [91]. However, type I collagen nanospheres have been shown to promote the osteogenic differentiation and proliferation of bone marrow stromal cells in vitro, thus highlighting their application in orthopedic treatments for bone tissue formation [92].

4. Challenges and future perspectives

Despite the vast potential of collagen in orthopedic diagnostics and therapeutics, there are certain challenges that hinder its widespread use. Further research is necessary to elucidate reliable markers of collagen metabolism that are indicative of particular disease conditions in order to increase the accuracy of collagen-based diagnostics. Simple, non-invasive, rapid detection techniques must be developed for assessing the levels of collagen and its metabolic products with a high degree of sensitivity and specificity in order to facilitate the use of collagen-based biomarkers in detecting orthopedic disorders like OA and RA [22]. The source of collagen for use in CBBs poses a serious problem—while collagen is abundantly present in most living organisms, its extraction from animal sources presents several risks including allergy, disease transmission, degradability, and immunogenicity. Collagen derived from bovine and porcine sources was seen to cause allergic reactions such as anaphylaxis in up to 4% of the population [59]. Additionally, prion-contaminated collagen from terrestrial animals can transmit zoonotic diseases like foot-and-mouth disease (FMD), transmissible spongiform encephalopathy (TSE), and bovine spongiform encephalopathy (BSE) which leads to the variant Creutzfeldt-Jakob disease (vCJD) in humans [93]. While these issues may be negated by the use of marine-derived collagen, the lack of substantial clinical trials coupled with its comparatively low thermal stability, rapid degradation in vivo, and poor mechanical strength impedes its application in orthopedics [94, 95]. Furthermore, animal-derived collagen telopeptides are antigenic and may trigger immune responses, leading to hypersensitivity reactions and graft vs. host diseases [96]. The use of collagen derived from human placenta and skin, on the other hand, is hindered by the large batch-to-batch variation in the biophysical properties of collagen based on parameters such as age, environmental factors, genotype, and ethnicity, which cannot be standardized [97]. Moreover, human-derived collagen presents ethical concerns related to informed consent, religious beliefs, and data privacy. Despite ongoing research into artificially synthesized collagen-like peptides, they cannot substitute natural collagen due to high costs and absence of the natural tertiary structure, due to which they cannot reproduce several biophysical properties necessary for biomedical applications [98]. Though it is considered a viable alternative, recombinant human collagen (rhCOL) also has certain shortcomings including the deficit of high protein throughput models, poor yield in mammalian cell cultures, high production costs, increased susceptibility to proteolytic degradation, higher frequency of misfolding in prokaryotic systems, and instability due to the lack of post-translational modifications in collagen obtained from expression systems that lack prolyl 4-hydroxyprolin (P4H), a heterotetramer enzyme necessary for the folding of collagen polypeptide chains to form triple helical structures [99, 100]. PH4-transduced plant systems like Nicotiana tabacum and Zea mays are currently preferred since they produce good yields of type I collagen [101]. Thus, despite the abundance of collagen, each of its sources has certain disadvantages that need to be addressed to optimize the cost-effective, large-scale production of collagen for orthopedic use.

Furthermore, the techniques employed for the extraction and cross-linking of collagen as well as the sterilization of CBBs, also cause certain issues. Fibrillar collagen, in its native form, is insoluble and resistant to proteolysis, which is why only type I collagen can be extracted in its original form from adult tissues. Thus, collagen is generally extracted in the form of hydrolyzed, soluble molecules through denaturing treatments using acids, alkalis, and proteolytic enzymes. However, the fibril-forming capacity of these molecules is lower than that of native collagen, especially due to the protease-mediated cleavage of terminal telopeptides [102, 103]. Further, the cross-linking of hydrolyzed collagen using chemical agents like glutaraldehyde and poly-epoxy compounds may leave behind toxic residual compounds, thus rendering the CBB unfit for human use [104]. While physical cross-linking methods such as dehydrothermal treatment and UV irradiation do not pose the risk of toxicity, prolonged exposure could lead to the partial or even complete degradation of collagen [105]. Thus, non-conventional cross-linking methods using enzymes like transglutaminase and natural, biocompatible cross-linkers like genipin must be further developed [106]. The fragility and thermal sensitivity of CBBs prevent sterilization via several common methods like autoclaving and electron beam irradiation with gamma rays since these approaches cause molecular degradation and structural alteration of the collagen scaffolds. Even milder approaches like β-ray irradiation and ethylene oxide sterilization cause some level of damage, and their use is restricted to specific types of CBBs [103]. Currently, ethanol treatment is used for physically cross-linked collagen, while immersion in peracetic acid is employed in the case of acellular collagen ECM. However, no known method has left the molecular structure of collagen unaltered post-sterilization [107, 108].

Moreover, the inability of traditional CBBs to regenerate the fibrillar structure of natural collagen leads to issues such as poor tensile strength and loss of biophysical properties. In recent years, modern technologies such as plastic compression, electrospinning, and 3D bioprinting have been introduced for the synthesis of highly ordered collagen matrices [109]. Plastic compression involves the rapid expulsion of water from cell-seeded collagen gels in order to synthesize strong, tissue-like collagen matrices without the need for chemical cross-linking and cell activity. In order to improve the scalability and reproducibility of plastic compression, the technique has been modified using upward fluid flow, which facilitates the synthesis of multi-layered scaffolds [110, 111]. Electrospinning is widely used for CBB synthesis since it allows the fabrication of collagen nanofibers with different morphologies that mimic the structural properties of native collagen in the ECM. However, despite the architectural versatility and biocompatibility of electrospun collagen fibers, the solubilization and processing of collagen prior to electrospinning leads to undesirable properties such as low elasticity, increased hydrophilicity, low mechanical strength, and high degradability [109, 112]. Near-field electrospinning, also known as melt electrowriting, is a modified electrospinning process that involves the extrusion of collagen fibers by the force of the electrical field followed by their immediate collection to avoid bending instabilities, which is achieved by reducing the air gap. The process allows precise control over individual collagen fibers, thereby facilitating the fabrication of tailored collagen microstructures and nanofibers with precise geometries. However, parameters like air gap distance, applied voltage, spinneret needle geometry, melt composition, and flow rate must be standardized prior to the widespread use of melt electrowriting [113]. 3D bioprinting is another highly advanced technique that produces hierarchical constructs via layer-by-layer deposition using collagen solutions as the bioink. The major disadvantage of this technique is the poor mechanical strength and stability of the CBBs, which can be overcome by using hybrid collagen-based bioinks containing substances like thrombin, hydroxyapatite, alginate, agarose, silk, and gelatin. Mechanical stability can also be improved by printing within a pre-formed mold, using thermo-reversible supports like gelatin slurries, increasing the density of the collagen bioink, and cross-linking the collagen fibers [106, 114, 115]. However, despite the advantages of modern fabrication techniques, they must be optimized through further research in order to promote the clinical use of CBBs in orthopedic therapy.

5. Conclusion

In conclusion, collagen is a unique component of the ECM with several desirable properties such as biocompatibility, biodegradability, and mechanical stability that can be harnessed for use in orthopedics. Given the involvement of collagen in the pathogenesis of orthopedic conditions, its metabolic fragments serve as biomarkers for the detection of diseases like ankylosing spondylitis and rheumatoid arthritis. With the development of efficient detection strategies and a deeper understanding of the mechanisms underlying disease pathogenesis, the potential of collagen-based diagnostics can be translated into widespread clinical applications. Since the invention of the catgut in 1881, several advanced CBBs including scaffolds and microstructures have been developed for orthopedic use. Innovative approaches are required to overcome the drawbacks associated with the different sources of collagen as well as the currently used extraction, processing, and sterilization methods. Further research into modern fabrication techniques like melt electrowriting and 3D bioprinting is of utmost importance for the integration of CBBs into orthopedic therapy. Despite its shortcomings, the unique characteristics of collagen place it at the forefront of biomedical research in the field of orthopedics.

Acknowledgments

The authors thank Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India, for providing the infrastructure required to complete this project. NM is grateful for the financial support received from the Indian Council of Medical Research (ICMR), Government of India, India (Project Number ITR/Ad-hoc/43/2020-21, ID No. 2020-3286) and the Global Innovation and Technology Alliance (GITA), Department of Science and Technology (DST), Government of India, India (Project Number GITA/DST/TWN/P-95/2021).

Conflict of interest

The authors declare no conflict of interest.