Application of collagen in bone regeneration

Abstract

At present, there is a significant population of individuals experiencing bone deficiencies caused by injuries, ailments affecting the bones, congenital abnormalities, and cancer. The management of substantial bone defects a significant global orthopedic challenge due to the intricacies involved in promoting and restoring the growth of fresh osseous tissue. Autografts are widely regarded as the “gold standard” for repairing bone defects because of their superior tissue acceptance and ability to control osteogenesis. However, patients undergoing autografts may encounter various challenges, including but not limited to hernia, bleeding, nerve impairment, tissue death. Therefore, researchers in regenerative medicine are striving to find alternatives. Collagen is the most abundant protein in the human body, and its triple helix structure gives it unique characteristics that contribute to its strength and functionality in various tissues. Collagen is commonly processed into various forms such as scaffolds, sponges, membranes, hydrogels, and composite materials, due to its unique compatibility with the human body, affinity for water, minimal potential for immune reactions, adaptability, and ability to transport nutrients or drugs. As an alternative material in the field of bone regeneration, collagen is becoming increasingly important. The objective of this review is to provide a comprehensive analysis of the primary types and sources of collagen, their processes of synthesis and degradation, as well as the advancements made in bone regeneration research and its potential applications. A comprehensive investigation into the role of collagen in bone regeneration is undertaken, providing valuable points of reference for a more profound comprehension of collagen applications in this field. The concluding section provides a comprehensive overview of the prospective avenues for collagen research, underscoring their promising future and highlighting their significant potential in the field of bone regeneration.

The Translational Potential of this Article. The comprehensive exploration into the diverse functions and translational potential of collagen in bone regeneration, as demonstrated in this review, these findings underscore their promising potential as a treatment option with significant clinical implications, thus paving the way for innovative and efficacious therapeutic strategies in this domain.

Graphical abstract

1. Introduction

In the human body, collagen accounting for about one-third of the body weight of protein tissue is the most abundant protein [1]. The extracellular matrix (ECM) is a substance devoid of cellular components that permeates all tissues, which forms the physical environment surrounding cells and plays a crucial role in providing structural support and facilitating cell signaling processes [2]. The collagen is derived from the tropocollagen molecule and serves as a vital constituent of the natural ECM in tissues. Tropocollagen serves as the fundamental building block of collagen proteins, which removes the N-terminal propeptide domain and its subsequent conversion into mature collagen (Fig. 1) [3]. The generation of biomarkers are generated from the N-terminal peptides produced by procollagen proteins [4]. Collagen, being the primary constituent of connective tissue, assumes a pivotal function in ensuring the preservation of its structural integrity. It exhibits a distinctive configuration, characterized by the intertwining of three peptide chains (α-chains) to create a resilient triple helix structure. Each chain showcases a distinct repetitive pattern (Gly-X-Y), with Gly denoting glycine, and X and Y typically representing proline and hydroxyproline [5]. This triple helix arrangement gives collagen its unique properties that contribute to its strength and functionality in diverse tissues, enhancing the structural integrity, flexibility, and ability to recover from stress in living organisms [6]. The formation of collagen fibers occurs when mature collagen molecules assemble and cross-link. Collagen molecules with triple helix structures self-organize into fibrils, which are then arranged in a regular pattern called the D-band [7,8]. The gap separating two successive collagen molecules is referred to as the 'gap zone' [9]. In addition, the collagen fibrils provide additional sites for the aggregation of hydroxyapatite (HA) crystals, guiding the growth of mineral crystals along the elongated axis of the fibers and resulting in an augmentation of particle sizes [10]. The formation of mineralized collagen occurs through the organized deposition of nano-HA onto the collagen inorganic matrix, representing a significant level of complexity within the natural bone structure [11]. Mineralized collagen establishes a nanoscale structural base that contributes to the exceptional mechanical and biological properties exhibited by bone [12].

Fig. 1.

Structural characteristics and sources of collagen (Created with BioRender.com.). ECM, extracellular matrix.

At present, there is a significant population of individuals experiencing bone deficiencies caused by injuries, ailments affecting the bones, congenital abnormalities, and cancer [[13], [14], [15]]. The management of substantial bone defects presents a significant global orthopedic challenge due to the intricacies involved in promoting and restoring the growth of fresh osseous tissue [16,17]. Autografts are widely regarded as the “gold standard” for repairing bone defects because of their superior tissue acceptance and ability to control osteogenesis [18]. However, patients undergoing autografts may encounter various challenges, including but not limited to hernia, bleeding, nerve impairment, tissue death; in more severe cases, systemic infections and insufficient blood circulation can ultimately lead to the failure of bone replacement [19]. Therefore, researchers in regenerative medicine are striving to find alternatives [[20], [21], [22], [23]]. Collagen is commonly processed into various forms such as scaffold, sponge, membrane, hydrogel and composite materials. In the process of promoting bone regeneration, materials have been widely studied and applied in bone tissue construction. The incorporation of collagen into polymers, metals and so on, promotes bone regeneration. Nowadays, collagen is becoming increasingly significant in the field of bone regeneration as a substitute material resulting from its exceptional compatibility with living organisms, affinity for water, minimal potential for causing immune reactions, adaptability, and capacity to transport nutrients or medications [[24], [25], [26]].

2. Main types of collagen

Up to this point, around 29 varieties of collagen have been recognized, and among them, Collagen type I (Col-I) is predominantly found in the ECM, specifically in tissues like tendons and bones [[27], [28], [29], [30]]. Collagen classified into categories such as I., II., III. identified using Roman numerals and distinguished by the presence of a minimum of one domain with three helices [31]. This article primarily introduces the main types of collagen (Table 1).

Table 1.

Main type of collagen. Col-I, collagen type I; Col-II, collagen type II; Col-III, collagen type III; Col-IV, collagen type IV; Col-V, collagen type V.

Collagen type	α Chαins	Molecular species	Tissue Distribution	References
Col-I	α1(I), α2(I)	[α1(I)]2α2(I) , [α1(I)]3	Tendons, bone,teeth, skin, lung, heart, and the vasculature	[32,33]
Col-II	α1(II)	[α1(II)]3	Cartilage, vitreous,cartilagenous zones of tendon, and intervertebral disc	[36]
Col-III	α1(III)	[α1(III)]3	Dermis, aorta, uterus, admixture in tendon, intestine, blood vessels, in the reticular connective tissue of liver, spleen and surrounding internal organs	[39]
Col-IV	α1(IV), α2(IV), α3(IV), α4(IV), α5(IV), α6(IV)	[α1(IV)]2 α2(IV)	Basement membranes	[45,46]
		α3(IV), α4(IV), α5(IV), α6(IV)
Col-V	α1(V), α2(V), α3(V)	[α1(V)]2α2(V), [α1(V)]3, α1(V)α2(V)α3(V)	Placental/embryonic tissue, dermis, bone, cornea and cell surfaces	[[50], [51]]

Collagen type I (Col-I), the primary collagen type found in animals, plays a vital role as an essential constituent of the ECM within bone cells [32]. It is primarily found in bones, teeth, tendons, skin, lungs, heart, and the vascular system. It consists of one collagen alpha 2 (COL1α2 (I)) and two collagen alpha 1 (COLα1 (I)) chains [33]. The occurrence of osteogenesis imperfecta can be attributed to dominant autosomal mutations in the genes responsible for encoding Col-I. Rare pathogenic genes are involved in post-translational modifications, processing, folding, cross-linking of Col-I as well as bone mineralization and osteoblast differentiation [34]. The progression of pulmonary fibrosis is associated with elevated levels of COLα1(I) secreted by alveolar macrophages [35].

Collagen type II (Col-II) is mainly distributed in cartilage, cartilagenous zones of tendon, vitreous, and intervertebral disc, comprising a trimeric assembly of three COLα1 (II) chains, showcasing the characteristic triple helical architecture [36]. Due to its unique molecular structure, Col-II has found extensive use in both the food and medicine. The degradation and metabolic disorders of Col-II in human articular cartilage may impact the occurrence and progression of osteoarthritis. When osteoarthritis occurs, the degradation of Col-II in cartilage may exceed its synthesis, leading to an increase in C-terminal terminal peptide of Col-II. Then, Col-II fragments can induce inflammation, up-regulate IL-1β, and lead to chondrocyte apoptosis [37]. Post-translational alterations are essential for the assembly of Col-II molecules and the arrangement of supramolecular fibrils, thereby supporting the functionality of Col-II while significantly influencing autoimmune reactions associated with rheumatoid arthritis pathogenesis [38].

Collagen type III (Col-III) is found in human skin, tendons, large blood vessels, fascia, uterus and bowel, consisting of three COLα1 (III) chains encoded by the COL3A1 gene [39]. It serves as a essential component in reinforcing the structural integrity and robustness of multiple organs., while also exhibiting various additional roles. Within tissues, the size of Col-III fibrils is comparatively smaller when compared to Col-I [40]. Both Col-I and Col-III are prominent subtypes detected in the pelvic tissues of females. While fibrous Col-I can provide high levels of tension to tissues, Col-III is commonly found with Col-I in soft tissues that require greater flexibility and expansion [41]. The regulation of fiber structure and biomechanical characteristics of cartilage tissue heavily relies on the pivotal role played by Col-III [42]. Mutations in the COL3A1 gene are associated with the development of Ehlers-Danlos syndrome (EDS), a genetic disorder characterized by vascular complications and considered to be rare and potentially life-threatening. Additionally, there have been reported cases of individuals with COL3A1 mutations presenting arterial aneurysms even in the absence of clear indications of EDS [43]. Homozygous mutant mice with inactivated Col3A1 gene exhibit a significantly reduced lifespan [44].

Collagen type IV (Col-IV), core components of the basement membrane, plays a pivotal role in establishing a fundamental network that provides support for both epithelial and endothelial cells. It serves as a crucial barrier between different tissue compartments [45]. Human genetic data consistently show that the single-gene form of cerebral small vessel disease is attributed to a coding mutation in the adjacent gene COL4A1 or COL4A2, which codes for α1(IV) and α2(IV) collagen [46]. The glomerular basement membrane is a vital component in the kidney's filtration barrier. The normal function of the glomerular basement membrane relies on the assembly of trimeric forms consisting of specific isoforms, namely α3(IV), α4(IV), and α5(IV) isoforms, within it [47]. Alport syndrome is a disorder characterized by genetic and phenotypic variations, affecting the basement membranes of glomeruli, cochlea, and eyes due to mutations in the Col-IV genes [48].

Collagen type V(Col-V) is distributed in placental/embryonic tissue, bone, dermis, cornea, heart and cell surfaces that is a regulatory fibril-forming collagen [49].There are multiple molecular isoforms of Col-V, which are formed by combining three distinct polypeptide α chains (α1(V), α2(V), and α3(V)). These isoforms include α1(V)2 α2(V), α1(V)3, and α1(V)α2(V)α3(V). Although Col-V is a minor component of the ECM, it still plays an important role [50,51]. Its insufficiency is linked to the reduction of corneal clarity and the occurrence of typical EDS, whereas its excessive expression is observed in cancer, granulation tissue, inflammatory conditions, atherosclerosis, as well as fibrosis affecting the skin, lungs, liver, kidneys, and adipose tissue [49]. Col-V regulates the size of heart scars after ischemic injury. The depletion of Col-V resulted in an increase in the size of post-infarction scar, accompanied by a deterioration in cardiac function [52].

3. Source of collagen

The utilization of collagen as a biomaterial were first introduced by and William Macewen and Joseph Lister in 1881, when they developed collagen-based sutures made from sheep intestines [53]. In 1956, collagen began to be used as a cell culture matrix for promoting cell growth [18], and it was not until 1993 that the first collagen-based bone implant obtained approval from the Food and Drug Administration (FDA) [54]. The selection of Col-I is influenced by the diverse array of available sources, each possessing its own inherent strengths and weaknesses.

Currently, there are many collagen preparations available, which can be obtained from animal tissues or sources other than animals [55,56] (Fig. 1). The orchestration of formation and maintenance in multicellular tissues of larger vertebrates is ascribed to the craftsmanship of collagen proteins. Therefore, investigation into collagen derived from the ECM of animal cells has become well-established and finds extensive applications in biomedical, medical, and healthcare products. For example, the accessibility of bovine-derived Col-I makes it readily available, and its excellent compatibility with living organisms, natural biodegradability over time, and minimal likelihood of triggering immune responses further enhance its appeal in various applications [57]. The triple helix domain of bovine and porcine collagen shares a high degree of similarity with human collagen, however, there are differences in the immunological properties of the terminal peptide region [58]. They don't usually trigger an immune response in most people, but can cause problems in those who are allergic to collagen [59]. Therefore, it is necessary to undergo consecutive negative skin tests before receiving bovine-derived Col-I treatment [60]. One of the major concerns is the potential transmission of zoonotic diseases such as foot and mouth disease and various forms of bovine spongiform encephalopathies. Among these, the most severe risk for humans is associated with transmissible spongiform encephalopathy [61]. The utilization of collagen derived from pigs and cows raises various apprehensions, including: (i) the potential for transmitting zoonotic diseases, (ii) the possibility of eliciting an immune response, (iii) the complexity and high cost associated with purification procedures, and (iv) religious restrictions observed in Hinduism, Judaism, and Islam [61,62]. The extraction of collagen from sheep tendons is widely accepted across various cultures and religions due to its compatibility. Additionally, it has a reduced susceptibility to infection caused by transmitting pathogens [63,64]. Vidal et al. demonstrated that protein hydrolysates and collagens obtained from sheep slaughter by-products possess antimicrobial and antioxidant properties. The findings suggest the potential application of sheep collagen in the development of nutraceuticals that promote human health [65].

The characteristics of equine collagen, in comparison to collagen from other mammals, include its remarkable similarity to human collagen and a slightly lower homology ranking than bovine collagen. In terms of amino acid composition of Col-I in vertebrates, its evolutionary distance is relatively low and conservation is high, resulting in a homology rate of 95 % [66]. It is generally believed that there is almost no risk of zoonotic transmission between humans and horses, and there have been no reports of immune reactions. However, due to religious restrictions on consuming horse meat and products based on horse collagen. It is still uncertain if horses can come into contact with alphaviral equine encephalomyelitis, a zoonotic infection transmitted by mosquitoes [67]. Nevertheless, the number of acute encephalitis cases reported each year has been very low in recent years and no epidemic situation has been reported. Therefore, considering horse by-products as one source for extracting medical-grade collagen can help avoid issues related to zoonotic transmission [61]. Receiving Col-I from rat tails is a convenient and feasible process, although it necessitates the implementation of an extensive purification protocol to mitigate potential contamination risks [56].

Marine collagen is an attractive choice due to its reduced likelihood of disease transmission in comparison to collagen derived from mammals. It also faces fewer religious restrictions [68]. Marine collagen demonstrates comparable compatibility and functionality to that of mammalian collagen, without any accompanying restrictions, as the gene sequences for collagen are typically conserved and cross-species similarities exist [69]. Additionally, marine collagen has lower immunogenic activity towards patients allergic to mammalian products, such as α-galactose antigen [70]. Collagen, specifically Col-I, has been obtained from marine organisms such as fish, as well as marine mammals like whales and invertebrates including sponges, jellyfish, starfish, squid, and sea urchins [71]. But the denaturation temperature of most marine collagen is comparatively lower than its mammalian counterpart. The observed discrepancy can be ascribed to factors such as a diminished concentration of imino acid residues, variations in body temperature, and fluctuations in environmental conditions [62].

Studies have utilized genetic engineering techniques to choose diverse host cells, encompassing genetically modified animals, plants, insect cells, bacteria, and yeast, for the production of recombinant collagen. Recombinant collagen exhibits a remarkable advantage that can to be tailored directly at the structural sequence level, thereby facilitating the adjustment of its assembly for predetermined effects [72]. Nevertheless, factors such as exorbitant expenses and limited yield pose constraints on its utilization, impeding the replacement of animal-derived collagen with recombinant alternatives [55].

4. Synthesis of collagen

Fibroblasts are primarily responsible for the synthesis of collagen, while collagen proteins demonstrate tissue specificity and can also be synthesized by endothelial cells, smooth muscle cells, and keratinocytes. While the regulation of collagen protein transcriptional activity is primarily determined by cell type, the expression can also be modulated by a variety of growth factors and cytokines [73]. Under normal circumstances, the level of collagen gene expression remains consistently low; however, upon entering the wound healing phase, there is an upregulation in collagen transcription rates to commence tissue restoration. Nevertheless, if not restored promptly and appropriately to baseline levels, excessive production may lead to abnormal scar formation and fibrosis [74].

The synthesis of collagen is a intricate procedure, encompassing the transcription of genes within the cellular nucleus to the creation of procollagen triple helix and fibrils. After undergoing transcription within the cellular nucleus, the pre-procollagen chains, which refer to a single polypeptide chain, are translated by ribosomes that are bound to the cell membrane. This particular chain comprises three primary structural domains: specifically, the α-chain, the peptide located at the amino-terminal end, and the peptide situated at the carboxy-terminal end. Within rough endoplasmic reticulum (RER), a trimer of pro-alpha chains combines to create a procollagen molecule featuring a triple helical structure. Throughout this procedure, the pre-peptide sequences are eliminated. Lysine and proline residues undergo hydroxylation. Glycosylation takes place on the propeptides, and disulfide bond formation occurs. Following these post-translational modifications, individual procollagen molecules are transported to the Golgi complex where they get enclosed within vesicles and subsequently released into the extracellular space [75,76].

Most collagen genes exhibit complex exon-intron patterns, varying from 3 to as many as 117 exons. In numerous instances, the presence of diverse mRNA variants can be observed due to the existence of multiple initiation sites for transcription, selective exon splicing, or a combination thereof. In addition to the process of splicing, the pre-mRNA also undergoes including capping at the 5′ end and polyadenylation at the 3’ end. The mRNA that is bound by ribosomes is then translated into pre-procollagen molecules, which are subsequently transported to the RER for post-translational modifications. The RER is where the three front chains of the procollagen molecule are combined into a triple helix structure. These initial precursors expand within the lumen of the RER with assistance from a signal recognition domain and are recognized by their corresponding receptor [9].

Pre-procollagen polypeptides undergo three major post-translational modifications within the RER. The N-terminal signal peptide is removed [77]. Depending on the collagen type, specific proline and lysine residues are enzymatically hydroxylated after translation [78]. The stability of the triple helix structure requires the formation of intramolecular hydrogen bonds in the presence of 4-hydroxyproline [74]. These amino acids are catalysed by 4-hydroxyproline, 3-hydroxyproline and hydroxylysine by collagen prolyl 4-hydroxylases, prolyl 3-hydroxylases and lysyl hydroxylases [79,80]. All enzymes require ferrous ions, 2-oxoglutarate, molecular oxygen, and ascorbate as essential coenzymes [81]. In the assembly of fibril-like collagens, approximately half of the proline residues are hydroxylated at position 4, and the extent of prolyl-hydroxylation varies across different species. Organisms inhabiting colder environments exhibit diminished levels of hydroxylation [82].

Upon the addition of hydroxyl groups to the residues of lysine and proline, selected hydroxyl groups undergo glycosylation with galactose and glucose on lysine. The glucose-galactose residue is transferred onto the hydroxyl group of hydroxylysine through the action of glucosyltransferase and galactosyl-hydroxylysine transferase [80]. As a result, procollagen is formed by the assembly of hydroxylated and glycosylated polypeptide chains from left-handed triple helices into right-handed coils, using a folding process similar to that of a zipper. Once processed and assembled, the triple helix molecules are encapsulated within secretory vesicles in the Golgi apparatus and subsequently released as propeptides into the extracellular space. Subsequently, depending on the specific collagen protein type, further processing occurs for the procollagen trimer after its secretion [74]. In general, collagen peptidases are responsible for cleaving the propeptides and removing the ends of procollagen. This process leads to the formation of tropocollagens [83], which then spontaneously assemble into fibrils at the surface of cells [84]. The assembly of these fibrils is regulated by incorporating Col-V and/or Col-XI, which still contain parts of their N-terminal propeptide. These components co-assemble with Col-I, Col-II, and III to form fibrils [85].

5. Degradation of collagen

Collagen, due to its tightly arranged helical structure, possesses unique mechanical properties that enable it to resist most protein hydrolysis attacks [86]. In addition, mineral deposits surround fibrous collagen found in teeth, bone, and calcified cartilage, enhancing their resistance to degradation caused by cells. Numerous molecular pathways have been identified to be implicated in both extracellular and intracellular breakdown of collagen. The degradation of collagen in extracellular pathways involves certain members from the matrix metalloproteinases (MMPs) and cysteine protease which possess the capability to enzymatically cleave collagen [87]. On the other hand, the intracellular pathway for collagen protein degradation involves internalization of relatively intact procollagen fibers and partially degraded fragments of collagen proteins within the ECM [88]. This process of internalization is accomplished by means of phagocytosis, macropinocytosis, or endocytosis facilitated by uPARAP/Endo180, subsequently leading to degradation via lysosomal cysteine proteases [89].

5.1. Extracellular pathways

5.1.1. 1MMPs

The MMPs constitute a family of proteolytic enzymes with distinct substrates but common structural features including catalytic domains and C-terminal hemopexin domains [90]. The active site is zinc-dependent and is highly conserved [91]. Collagenases encompass MMP-1, MMP-8, MMP-13, and MMP-18 that cleave various ECM proteins as well as soluble proteins. But the primary function of these MMPs is to specifically cleave fibrillar collagen into two distinctive fragments: a 1/4 C-terminal fragment and a 3/4 N-terminal fragment. The process unfolds through a biphasic progression. Initially, MMPs initiate the unwinding process of collagen's triple helical structure. Subsequently, they catalyze the hydrolysis of peptide bonds [92]. The hemopexin domain plays a pivotal role in cleaving native fibrillar collagen [93].

Gelatinases, including MMP-2 and MMP-9, possess the capacity to degrade Col-IV, V, VIII, X and XI by virtue of their three repeat fibronectin type II domains that facilitate binding to denatured collagen. While primarily functioning as a gelatinase, MMP-2 also exhibits weak collagenase activity [94]. MMP-2 degrades collagen through two step process. Firstly, MMP-2 induces weak interstitial collagenase-like collagen degradation, and subsequently facilitates gelatinolysis through fibronectin-like domains [90]. MMP-9 could function like a collagenase and gelatinase, that has degraded collagen more efficiently compared to other collagenases MMPs [95].

Within the MMPs, membrane-type MMPs(MT-MMPs) constitute a distinct subclass that is characterized by their anchorage to the cellular membrane via a single transmembrane domain. These MT-MMPs possess structural domains similar to soluble matrix metalloproteinases, including characteristic catalytic domains and C-terminal hemopexin domains. The presence of this transmembrane domain is essential for the enzyme to be localized at the cellular surface [96]. Before collagen fibrils are cleaved, MT1-MMP forms homodimers through the hemagglutinin domain. This dimer plays a crucial role in the cleavage of Col-I fibers and acts as a suppressor of homodimer formation [97].

5.1.2. The role of MMPs and their inhibitions in bone regeneration

It has been established that MMPs are involved in a diverse range of metabolic processes, including bone regeneration, tissue repair, cell proliferation, wound healing, programmed cell death, and gonadal tissue regeneration [98]. MMPs activity and overexpression lead to a variety of bone diseases, including osteoporosis, arthritis, osteonecrosis, or bone cancer metastasis [99,100]. It has been reported that local intervention of bone defects using the molecular MMPs inhibitor Marimastat holds promise as a potential therapeutic strategy for enhancing bone regeneration in individuals with diabetes [101].

However, the utilization of animal models exhibiting MMPs deficiency has elucidated a significant association between MMPs and bone homeostasis, which is evident in various bone anomalies such as delayed ossification and fracture repair, aberrant bone development, irregular bone morphology, or diminished fracture resistance [102,103]. Furthermore, certain MMPs possess the ability to interact with the collagen matrix and govern the process of collagen remodeling. Arai et al. related that the absence of MMP-13 results in a reduction in osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on the Col-I matrix. In addition, pretreatment with recombinant human MMP-13 leads to the remodeling of Col I matrix and promoted osteogenic differentiation and in vivo bone formation of hMSCs by up-regulating the expression of osteogenic genes runt-related transcription factor 2 (RUNX2), integrin α3 (ITGA3) and plaque adhesion kinase. In addition, the transcription factor RUNX2 binds to the MMP-13 promoter. These results suggest that MMP-13 grows on the modified Col I matrix and stimulates osteogenic differentiation and bone tissue self-healing of hMSCs through a positive MMP-13/ITGA3/RUNX2 feedback loop [104]. MMP-cleavable peptides, comprising a specific amino acid sequence, exhibit sensitivity to various MMPs. These peptides mimic the ECM and can be recognized and degraded by MMPs at the cleavage site [105]. Zhang et al. successfully constructed a MMP-responsive injectable hydrogel for precise drug release upon inflammation stimulation. This innovative approach allowed for a substantial drug release during the early stage of inflammation, while maintaining sustained drug release throughout the later stages of bone repair. In vivo and in vitro experiments, hydrogel had the capacity to induce macrophage polarization into the anti-inflammatory M2 phenotype, thereby promoting osteogenic differentiation and facilitating new bone regeneration [106].

In summary, since the ECM is a dynamic structure, the overexpression, inhibition and deficiency of MMPs can affect the remodeling of the ECM. The ECM would be altered by altering the activity of MMPs, while overexpression of MMPs or gene defects can lead to the failure of the bone repair process, and regulating MMPs expression remains a great challenge.

5.1.3. Cathepsin K

Some forms of collagen can also be broken down by lysosomal cysteine proteases. When cells are exposed to specific inhibitors of these proteolytic enzymes, the involvement of cathepsin in collagen degradation becomes apparent. It has been reported that tissue cathepsin B and L possess collagen-degrading abilities, as they have the capability to cleave non-helical extensions found on collagen fibrils [107,108]. Cathepsin K is a papain-like cysteine protease that is mainly expressed in osteoclasts, essential for Col-I and elastin matrix recycling [109]. MMPs cleave the collagen triple helix, but they neither clean collagen efficiently or degrade the cross-linked pyridine-deoxypyridinoline telopeptide that forms the terminal region of the collagen triple helix. In contrast, cathepsin K efficiently clefts collagen triplex helices and telopeptides, resulting in collagen monomers. Cathepsin K could completely dissolve human cortical bone collagen in vitro [109,110]. It is capable of breaking down tight collagen fibers into fragments and further dissolving them into soluble peptides through structuring as elongated c-shaped enzymes which bind to the glycosaminoglycans coating on collagen fibrils. Upon binding with glycosaminoglycans, cathepsin K breaks down both ends and multiple sites along the triple helix structure of collagen fibrils [111].

5.1.4. The role of cathepsin K and their inhibitions in bone regeneration

In addition to its high expression in osteoclasts, cathepsin K is also detected in various tissues and cell types, encompassing adipose tissue, dermis, myocardium, lung, smooth muscle cells, ovary, placenta, thyroid, liver, macrophages, cartilage, osteoblasts and osteocytes, breast and prostate cancers [112]. The defective cathepsin K propeptide and mutations in the polypeptide chain of the mature enzyme have been demonstrated to impede proper folding, resulting in impaired collagen binding and decomposition. This subsequently leads to bone dysplasia characterized by compromised bone remodeling, ultimately bring to bone sclerosis and an increased susceptibility to fractures. The radiological assessment reveals acro-osteolysis and high bone density, which are characteristic manifestations of this disorder [113,114]. Zhang et al. reported that cathepsin K deficiency promoted alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSCs). This finding was substantiated by RNAseq data, indicating that inhibition or knockdown of cathepsin K enhanced JBMMSC regeneration through glycolysis [115].

While currently available antiresorptive drugs reduce osteoclast activity and number, resulting in reduced bone resorption and secondary reduction in bone formation. Cathepsin K inhibition results in increased osteoclast number, which remain on the bone surface despite impaired ability to take up bone matrix. Then they appear to be able to send local signals to neighboring cells, including osteoblasts and osteoblast lineage cells. Thus, the function and activity of osteoblasts are maintained [112]. Nowadays, cathepsin K is a potential anti-resorptive drug target for the treatment of osteoporosis. Although there are no FDA-approved drugs, several generations of cathepsin K inhibitors have been developed, such as Relacatib, Balicatib and Odanacatib. Lessons learned about the potential biological mechanisms and clinical efficacy of feline peptidase K inhibitors, as well as adverse events, should continue to guide future drug development efforts to treat bone defect related diseases.

5.2. Intracellular pathways

Several pathways of collagen uptake and intracellular degradation are as follows: (i) Phagocytosis is initiated when members of the β1-integrin family recognize collagen fibers and interact with non-collagenous proteins covering their surface. This recognition triggers pseudopods rich in actin to engulf a portion of the collagen fiber, resulting in cleavage and internalization of a fragment. The initial breakdown of the fiber requires membrane-bound MT1-MMP [116]. (ii) Cells utilize macropinocytosis to internalize fragmented collagen fibers found in extracellular spaces. Actin-mediated uptake leads to the degradation of soluble collagen protein fragments within large protein bodies formed during this process, facilitated by cysteine proteases [117]. (iii) The receptor-mediated endocytosis involves crucial roles played by uPARAP and megalins as receptors. Upon uptake mediated by uPARAP, cysteine proteases degrade the internalized collagen fragments [[118], [119]]. (iv) Autophagocytosis represents a distinct degradation pathway where double-membrane-bound autophagosomes envelop cytoplasmic fragments containing double-membrane-bound organelles derived from endoplasmic reticulum that contain collagens segments. After undergoing fusion with lysosomes, the collagen segments undergo degradation by cysteine proteases within autolysosomes [120].

6. The role of collagen in bone regeneration

In the human organism, the skeletal system belongs to a family of tissues characterized by intricate arrangement. The skeleton consists primarily of HA, a compound rich in calcium phosphate, along with collagen protein and various additional constituents such as water and proteins [121]. Bone repair involves a series of precise and coordinated biological processes, which occur in three consecutive stages: inflammation, repair, and remodeling (Fig. 2). These stages involve the participation of various cells and intercellular signaling pathways. In the normal bone healing process, the inflammatory stage is the initial phase following a fracture. During this stage, there is local vasodilation and leakage of plasma and leukocytes leading to hematoma formation [122]. Sequential recruitment of immune cells such as neutrophil granulocyte, macrophage, and lymphocyte occurs to secrete cytokines like interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), receptor activator for nuclear factor kappa B ligand (RANKL), angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF). These cytokines promote migration of endothelial cells into the hematoma to facilitate new blood vessel formation. This process provides nourishment for subsequent migration events and aids in stem cell migration [123].

Fig. 2.

Bone repair involves three stages: inflammation, repair, and remodeling (Created with BioRender.com.). IL-1interleukin-1; TNF-α, tumor necrosis factor-α; RANKL, receptor activator for nuclear factor kappa B ligand; Ang-1, angiopoietin-1; VEGF, vascular endothelial growth factor; MSCs, mesenchymal stem cells; Col-I, collagen type I; Col-II, collagen type II; Col-IV, collagen type IV; Col-IX, collagen type IX; Col-X, collagen type X; Col-XI, collagen type XI.

During the repair phase, fibroblasts and mesenchymal stromal cells (MSCs) migrate to the bone defect site from both peripheral blood and surrounding tissue. Vascular buds grow into the affected area and granulation tissue is formed along with the deposition of collagen matrix. The formation of granulation tissue involves two processes: intramembranous ossification and endochondral ossification. Intramembranous ossification takes place on the periosteum and directly leads to the development of solid granulation tissue. Osteoblasts are derived from MSCs located between the periosteums, which undergo proliferation and differentiation to form osteoblasts, directly contributing to the formation of woven bone. Soft granulation tissue is formed through endochondral ossification within both bone and bone marrow before it transforms into hard granulation tissue. Chondrocytes, differentiated from MSCs in the bone marrow, secrete cartilage matrix to establish a template made of cartilage. The chondrocytes then undergo hypertrophy differentiation, causing mineralization of surrounding matrix to form cartilaginous granulation tissue. Ultimately, hypertrophic chondrocytes experience programmed cell death, resulting in the infiltration of blood vessels and the movement of osteoblasts. This is followed by a conversion of cartilage matrix to bone matrix [124].

During the final phase of bone healing, bones undergo a remodeling process aimed at restoring their initial structure, shape, and mechanical characteristics. The interplay between osteoblasts and osteoclasts is pivotal in regulating the deposition and resorption of lamellar bone throughout the bone remodeling procedure [125]. Cartilage serves as the precursor to bone, a resilient and pliable tissue devoid of blood vessels. Over time, it undergoes a physiological transformation known as endochondral ossification, gradually transitioning into rigid bone tissue. The ECM of articular cartilage has a highly organized structure, which is maintained by chondrocytes in response to mechanical loading. Chondrocytes are embedded within the matrix along with extracellular fibers [126]. In addition to the predominant Col-I, there are various minor collagens present in cartilage that perform different functions depending on their location within natural bone tissue [6]. For instance, instead of Col-I, the primary solid constituent in cartilage tissue's transparent matrix is Col-II. Its role involves preserving the overall form and arrangement of cartilage by restricting the expansion pressure exerted by proteoglycans with negative charges. Furthermore, Col-VI can be observed in the extracellular matrix that surrounds chondrocytes, whereas minimal quantities of Col-IX and Col-XI are present in the intercellular matrix. Col-IX is found within the pericellular matrices of chondrocytes and interacts with Col-II, thereby contributing to the biomechanical properties of bone tissue. Col-XI is responsible for regulating the thickness of Col-II fibers. It is worth noting that Col-X exclusively exists at the interface between calcified cartilage and subchondral bone contact area [31]. Considering that successful bone regeneration requires integration of live cells, growth factors and cytokines with ECM materials possessing good biocompatibility for osteoconduction and osteogenesis processes. Particularly Col-I provides an excellent material for bone tissue engineering (BTE) due to its ability to improve sufficient mechanical strength as well as vascular environment for osteogenic induction.

Throughout the ossification process, osteoblasts release Col-I and various non-collagen proteins like osteocalcin, osteopontin, and bone sialoprotein. The ECM initially secreted by osteoblasts may lack structure and crystallinity but gradually transitions into a more crystalline state [10]. Mineralization is facilitated by osteoblasts to promote bone formation and is thought to begin with the formation of matrix vesicles that bud from these cells' plasma membrane. This creates an environment suitable for calcium and phosphate ions to concentrate, promoting crystal growth. Collagen fibers also serve as additional sites for HA crystals to nucleate and aggregate, guiding their growth along the fiber axis resulting in larger particle sizes [8].

Mineralized collagen represents a significant level within the intricate hierarchical arrangement found in the composition of natural bone, as it involves the ordered deposition of nano-HA onto a collagenous inorganic matrix [127]. Mineralized collagen provides a nanoscale foundation for excellent mechanical and biological properties in bones [128]. Depending on how hydroxyapatite is distributed relative to collagen fibrils, mineralized collagen can be classified into intrafibrillar mineralized collagen or extrafibrillar mineralized collagen. Intrafibrillar mineralized collagen refers to mineral deposition occurring within the collagen protein matrix, while extrafibrillar mineralized collagen involves random mineral deposition on its surface [129]. Finally, certain osteoblasts find themselves enclosed within the bone matrix they contributed to creating. These particular cells are known as osteocytes.

Collagen promotes bone defect repair and regeneration through the following mechanisms: it stimulates osteogenic differentiation of bone MSCs, inhibits osteoclast differentiation in mineralization formation [130], transmits signals via integrin or non-integrin receptor cell surface stimulation of collagen protein, promotes fibroblast proliferation, leading to fibroblasts, endothelial cells and inflammatory cells migrating to the wound site while reducing axonal degeneration and disintegration [57].

7. Applications of collagen in BTE

Collagen has the potential to undergo various physical transformations and can be utilized with a diverse range of manufacturing techniques. Collagen-derived substances in diverse formats, such as scaffold, membranes, hydrogel, sponges and micro- and nanospheres (Fig. 3), are extensively employed within living organisms to facilitate the restoration of bone tissue in various medical scenarios.

Fig. 3.

Collagen-derived substances in diverse formats (scaffold, membranes, hydrogel, sponges and micro- and nanospheres) are prepared by compositing with natural polymers, synthetic polymers, metals and inorganic non-metallic materials in bone tissue engineering (Created with BioRender.com.). PCL, polycaprolactone; PLGA, poly (lactic-co-glycolic acid; PVA, polyvinyl alcohol; HA, hydroxyapatite; β-TCP, β-Tricalcium Phosphate.

7.1. Scaffold and composites

The arrangement of bone tissue's structure plays a crucial role in BTE, as it impacts both mechanical functionality and biological reaction [131]. In order to fulfill the load-bearing function of natural bones, scaffolds should also promote vascular formation. The scaffold's interconnected porous architecture facilitates the migration and proliferation of endogenous cells. Furthermore, the design optimization should not only ensure an adequate surface area for cell–scaffold interactions but also facilitate efficient diffusion of oxygen, nutrients, and waste removal. Typically, pore sizes ≥300 μm are required to promote new bone and blood vessel formation, with an acceptable minimum size being ≈100 μm [132]. Larger pore dimensions have been demonstrated to be the favored option for facilitating bone growth as they offer sufficient room for oxygen and nutrient provision, thereby enhancing vascularization within recently developed osseous tissue [133].

In previous studies, researchers have explored different types of scaffolds for tissue and organ regeneration, including those made from natural or synthetic polymers like collagen or chitosan. These materials were chosen due to their exceptional compatibility with living organisms, ability to break down naturally over time, suitable mechanical characteristics, and presence of porous structures [6]. In 1993, Collagraft™, the initial bone implant composed of bovine collagen mixed with the patient's own marrow and combined with HA/tricalcium phosphate (TCP), received FDA approval. Bongold™ is a patented composite material consisting of Col-I and synthetic HA. When assimilated with the host's bone marrow aspirate, it becomes both osteoconductive and osteogenic [134]. The mineral composition of this product is approximately 50 % less than that of natural bone, without any substitution of carbonate. It is exclusively available as a filler for cancellous bone voids and comes in cylindrical, block, and granular forms to accommodate various cavity sizes. Bongold™ is also marketed under the brand names HEALOS®, OssiMend®, Vitoss®, and Mastergraft®. Extensive research has demonstrated that this product line serves as a viable alternative to autografts in procedures involving fixation of metal implants [135]. SynOss™ is another notable calcium phosphate-collagen composite material with similar distribution forms as Bongold™ but specifically designed for dental applications [136]. The main difference between these two products lies in their composition; while both contain synthetic hydroxyapatite, SynOss™ has a higher concentration of carbonate minerals at almost double the amount (80 % compared to 45 %) Despite having a composition closer to that of bones, studies have reported that SynOss^TM fails to induce bone regeneration in most cases when used as scaffolding [137]. Toosi S et al. conducted a study nvestigating the efficacy of collagen and polyglycolic acid (CPGA) stents combined with bone marrow mesenchymal stem cells (BMSCs) for treating scaphoid nonunion. Their findings suggested that CPGA and cell therapy held promise as a potential alternative to bone grafting in addressing bone nonunions [138]. Apatzidou et al. assessed the safety and effectiveness of utilizing collagen scaffolds to implant BMSCs for periodontal reconstruction. Clinical and radiographic evaluations were performed both before anesthesia and postoperation at regular intervals over a span of 12 months. Prior to the transplantation of the biocomplex, all quality controls were successfully met. Notably, no adverse healing events were observed during the course of the study [139]. In a study of Sotome S et al., patients who had bone defects resulting from benign bone tumors, fractures, or autograft harvesting were randomly assigned to receive either porous HA/Collagen or porous β-Tricalcium Phosphate (β-TCP) implants. The researchers observed that when it comes to promoting bone regeneration, there are certain benefits associated with using porous HA/Col as opposed to using porous β-TCP. Nevertheless, it should be noted that the occurrence of adverse reactions was relatively higher among individuals who received the porous HA/Col implants compared to those who received the β-TCP implants; however, no severe rejection reactions were reported [140]. According to the findings of Kawai et al., the initial utilization of a composite material consisting of octacalcium phosphate and collagen was observed in treating bone defects in human subjects [141]. Filardo et al. conducted a study on patients suffering from symptomatic knee osteochondritis dissecans and treated them with the implantation of a scaffold made of collagen-hydroxyapatite, which mimics natural bone tissue. The clinical outcomes after 2 years were found to be promising, regardless of the size of the lesion. This suggests that even large lesions can benefit from this innovative implant. However, less favorable results were observed when using Magnetic Resonance Imaging (MRI) techniques for evaluation purposes [142].

7.2. Membranes

Collagen membranes that can be absorbed have been utilized for the purpose of directing tissue and bone regeneration procedures, owing to their established biocompatibility and capacity to facilitate wound healing. Successful membrane material design should also consider the following characteristics: (i) Biocompatibility, which ensures no harm to surrounding tissues and promotes healing processes; (ii) Cell occlusion, preventing non-osteogenic cells from invading bone defects through the mucosa; (iii) Ease of operation, with a balance between hardness and spatial maintenance functionality [143]. They possess a diverse range of porosity, facilitating the selective migration of cells and the transmission of chemicals, biomolecules, and viruses. In general terms, the pore size varies from microporosity (5–20 μm) to moderate porosity (≤100 μm for non-absorbable materials that allow for integration/migration of bacteria, cells, and tissues at ≥30–40 μm) to large porosity (>100 μm for non-absorbable materials that enable unhindered passage of chemicals, biomolecules, viruses, bacteria, cells as well as integration and migration of tissues). The pore size may expand during membrane degradation process and impact its sealing functionality [144,145]. In the field of oral surgery, collagen barrier membranes are commonly used for regenerating periodontal defects as they possess the advantageous characteristic of being biodegradable. The initial batch of membranes, comprising of non-absorbable materials like expanded polytetrafluoroethylene and high-density polytetrafluoroethylene [146], gained significant popularity during the 1990s and are widely acknowledged as the benchmark in this field [147]. While membranes of the first generation exhibit satisfactory spatial maintenance, they are accompanied by significant drawbacks including heightened rates of exposure and bacterial infection. Consequently, an additional surgical procedure is often required to eliminate the membrane [148]. To address these challenges, a promising solution lies in the utilization of absorbable collagen membranes which offer numerous benefits such as simplified surgery process, enhanced healing of soft tissues, consolidation of host tissues with membranes, and swift absorption in case of exposure [149]. However, the disadvantages of using collagen protein membranes in guided tissue regeneration are: (i) loss of space-maintaining ability under wet conditions; and (ii) potential risks associated with implanting animal-derived collagen, including disease transmission from animals to humans [8].

Numerous collagen membranes with distinct chemical and physical structures have been created and are currently employed in clinical settings, including Bio-Gide®, Collprotect®, Jason®, and Ossix®. These variations arise from the utilization of diverse collagen sources and extraction techniques. The primary commercial collagen membrane, Bio-Gide®, is comprised of Col-I and Col-III sourced from porcine phosphoric acid. It features a dual-layer composition consisting of a compact layer and a permeable layer that facilitates controlled barrier functionality [150]. This dual-layer structure promotes the migration and specialization of osteoblasts while inhibiting fibroblast infiltration, making it an established solution for designing barrier membranes. The compact layer of Bio-Gide® remains intact for 60 days post-implantation, whereas the permeable layer undergoes complete degradation [143]. In their study, Rothamel et al. investigated the compatibility of different collagen membranes with cultured human periodontal ligament fibroblasts as well as human osteoblast-like cells. Their findings revealed that Bio-Gide®, followed by Ossix®, exhibited a notably higher level of adherence from periodontal ligament fibroblasts compared to Tutodent®, while all three showed superior adherence in comparison to Biomend®. Moreover, when it came to osteoblast cell adhesion, both Bio-Gide® and Tutodent® outperformed Ossix®, whereas no presence of osteoblast-like cells was observed on the Biomend® membrane [151].

7.3. Hydrogels

Hydrogels are polymer materials that swell in water and are created by cross-linking reactions to form a network structure [152]. In comparison to synthetic or hybrid polymers, hydrogels derived from collagen of natural origin demonstrate superior biocompatibility and bioactivity [153]. However, the physicochemical and mechanical properties of collagen hydrogels are not ideal and they tend to shrink significantly during cell culture. This limitation hinders their potential applications in BTE. As a result, researchers often combine collagen with other polymers or active inorganic substances to create hydrogels that can provide an environment conducive to bone growth for stem cells and expedite the regeneration of bone tissue [6].

i-Factor™ is a biocompatible hydrogel carrier containing anorganic bone mineral suspended in it, with P-15 synthetic collagen fragment adsorbed onto it as a composite bone substitute material. Arnold PM et al. conducted a study where participants were assigned to receive either autograft or i-Factor™ in a cortical ring allograft. The researchers then assessed the outcomes using radiological, clinical, and patient-reported measures. The results indicated that at the 2-year follow-up after surgery, i-Factor™ demonstrated comparable efficacy to local autograft bone [154]. Alkimavičienė E et al. evaluated the effectiveness of collagen hydrogels and proanthocyanidins (PACNs) as an additional treatment for patients with periodontitis. The inclusion of PACNs in conjunction with minimally invasive nonsurgical therapy demonstrated enhanced improvement in MMP-3 concentration in saliva [155]. CaReS® is a technique that employs Col-I hydrogel-based autologous chondrocyte implantation for addressing osteochondral defects in the knee. Rackwitz et al. showed that the CaReS® technique proved to be both clinically effective and safe for reconstructing isolated osteochondral defects in the knee joint, showcasing promising clinical outcomes even up to 5 years post-surgery [156]. Collagen hydrogels have also been used as hemostatic agents. It is approved by FDA for biomedical use that is now commercially available. A practical example is VITAGEL®. Its application in surgical procedures is primarily utilized when traditional methods of managing bleeding prove to be ineffective or impractical. VITAGEL® promptly adheres to the patient's own plasma prior to its administration at the site of bleeding. Rapid formation of fibrin/collagen clots effectively regulates hemorrhaging and facilitates a three-dimensional matrix that fosters the process of healing [8].

7.4. Sponges

Due to their exceptional functionalities and properties along with convenient processing methods plus effective sterilization techniques [157], collagen sponges have emerged as highly valuable biomaterials. The formation of collagen sponges typically involves a freeze-drying procedure (also referred to as ice crystal template technique or ice separation-induced self-assembly) [158]. Altering freezing parameters like temperature duration and mold enables customization of pore size and shape in collagen sponges. To ensure optimal tissue regeneration through enhanced biological activity, it is crucial that pores allow for both cell migration/nutrient diffusion while facilitating cell attachment. In BTE, collagen sponges primarily function as foundational scaffolds capable of carrying various bioactive agents such as growth factors cells drugs, etc [4]. However, due to the limitations of pure collagen sponges such as poor mechanical properties, insufficient osteoinductive activity, and easy degradation, modified or composite materials with inorganic substances are commonly utilized for BTE composites. The incorporation of FDA-approved recombinant human bone morphogenetic protein-2 (rhBMP-2) into absorbable collagen sponge has been found to enhance bone formation. Collagen-targeted rhBMP-2 exhibits strong affinity towards collagen proteins and stable binding over a long period of time while also inducing new bone formation even at low concentrations compared to natural rhBMP-2. Therefore, utilizing collagen sponge composites may offer potential for developing third-generation implants that actively support vascularization and promote bone tissue regeneration leading to improved patient care [159].

7.5. Microspheres and nanoparticles

Nanoparticles are a promising drug carrier in the field of nanoscale drug delivery systems due to their ability to regulate the release of drugs, enhance cell membrane permeability, modify biological distribution, and improve drug bioavailability [160]. Microspheres, on the other hand, possess a three-dimensional spatial structure and act as porous spherical materials. They offer an increased surface area for cell growth and adhesion while facilitating diffusion and mass transfer behavior that can be easily estimated [161]. In biomedical applications like tissue engineering, bone transplantation, and drug delivery encapsulation materials, microspheres have gained significant popularity. Collagen or collagen/polymer-based microspheres, liposomes, or vesicles have been employed to facilitate cellular proliferation on scaffolds. Specifically in the realm of bone tissue regeneration, collagen particles can be modified to incorporate various bioactive substances such as growth factors (e.g., BMP, VEGF), cells (e.g., MSCs, osteoblasts, osteoclasts, human umbilical vein endothelial cells), and medications (e.g., antibiotics). In the field of BTE, collagen microspheres and nanoparticles are commonly integrated into scaffolds composed of synthetic polymers and/or bioceramics (such as HA) with the objective of enhancing osteoblast growth within bone fillers [162].

8. Collagen with composite components applications in BTE

In the process of promoting bone regeneration, materials have been widely studied and applied in bone tissue construction [163]. The incorporation of collagen into natural polymers such as alginate, chitosan, and hyaluronan, etc, and synthetic polymers including polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (PVA) and so on, promotes bone regeneration [6]. The utilization of metals and their alloys as biomaterials for bone repair is attributed to their exceptional mechanical strength and fracture toughness. Inspired by this strategy, adding metals and their alloys into collagen to prepare composite scaffolds can significantly enhanced the corrosion resistance and mechanical strength of the composite materials [164]. Inorganic bioactive components, such as bioceramics which are similar to the inorganic components of natural bone fixation, is widespread in the field of bone regeneration [165,166].

8.1. Polymers materials-based composites

8.1.1. Natural polymers

Natural polymers provide a suitable bioactive environment for cellular activity, minimal adverse immune reactions, and essential mechanical reinforcement to facilitate the proliferation of new osseous tissue at the site of defect. In the past, as an important structural material, natural polymer materials, such as alginate, chitosan, silk fibroin, and hyaluronan, have been extensively used in BTE.

Alginate, derived from algae, possesses abundant sources, low cost, excellent biocompatibility and biodegradability, non-toxicity as well as remarkable performance in scaffold formation. However, the absence of the Arg-Gly-Asp (RGD) -binding motif leads to diminished cellular adhesion [167]. Alginate-mediated collagen assembly and synthesis has been reported in the literature. Alginate-mediated effect includes transverse aggregation of small collagen fibers into huge bundles to produce an exquisite 3D mineralization structure with enhanced mechanical properties, and the upregulation of transcription factors associated with osteogenic differentiation is higher during alginate-assisted collagen mineralization [168]. Chitosan, a deacetylated polysaccharide from the shell of Marine crustaceans, has low immunogenicity, good biocompatibility, biodegradability, low bioabsorbability, and low economic feasibility, and is a potential candidate material for regenerating BTE. But the application of Chitosan scaffolds in BTE is limited by their biological activity and mechanical properties [169]. It has been reported that an optimized bio-composite scaffold, composed of chitosan/Col-I/HA, which had a highly porous structure. The MC3T3-E1 proliferation, osteogenic-related gene expression, and matrix mineralization were significantly enhanced in the presence of collagen, with a more pronounced effect observed as the amount of Col-I increased [170]. Hyaluronan is found in body fluids and tissues, which is another important component of the ECM. It exhibits a diverse range of physiological and structural functions, encompassing cellular interactions, growth factor signaling, and osmotic pressure regulation. Collectively, these multifaceted roles contribute to the maintenance of organizational integrity and homeostasis [171]. But due to its water-soluble properties and enzymatic degradation properties, it cannot be matched to the tissue repair process, which hinders its intended application in BTE [172]. A 3D printed polymer partial meniscus scaffold was successfully fabricated composed of collagen and hyaluronan, that could generate functional meniscus tissue with an instantaneous compression modulus of 109 % of natural sheep meniscus, in addition to improving the contact mechanics of the knee joint without adversely affecting the surrounding articular surface [173]. Silk fibroin is a natural material widely used in the pharmaceutical and textile industries. It is extracted from domestic silkworm cocoons, good cytocompatibility, that has been used in various tissue engineering applications [174]. Liu et al. showed silk fibroin and collagen were blended in a weight ratio of 75:25 to fabricate membranes that exhibited excellent biocompatibility, favorable mechanical properties in the wet condition, and appropriate biodegradation rate. Furthermore, the hybrid biological functions of silk fibroin and collagen synergistically enhanced MC3T3-E1 osteoblast cell adhesion and proliferation on the membranes [175].

8.1.2. Synthetic polymers

In addition to natural polymers, synthetic polymers such as PCL, PLGA, PVA et al. have also been widely used in BTE. Natural polymers exhibit biodegradability and good biological activity, which partially address the mechanical performance deficiencies. But specific applications in BTE require certain modifications to the polymer structure. The reengineering process poses significant challenges when dealing with natural polymers, whereas it is comparatively straightforward for synthetic polymers [165]. Synthetic polymers exhibit good mechanical properties, biodegradability, and ease of processing. However, the inadequate cell adhesion, limited biological activity, and hydrophobic nature of these materials restrict their application as bone repair substitutes. To overcome these limitations and fully exploit their advantages, numerous scholars have extensively explored collagen-based synthetic polymer composites to meet the criteria of bone graft substitutes [176].

PCL is an FDA-approved synthetic polymer commonly used in a variety of BTE applications, which is characterized by its aliphatic nature, semi-crystalline structure, and remarkable combination of toughness, mechanical strength, and biocompatibility. While it's slow degradation rate sometimes poses as a factor that impacts negatively on BTE process. Besides, the hydrophobic nature of PCL inhibits cell adhesion and consequently impedes proliferation [177]. Fibrous membranes for guided bone regeneration have been successfully fabricated by using electrospinning technique comprising PCL, Col-I and mineralized Collagen. Moreover, the membranes demonstrated exceptional water absorption capability while maintaining their original thickness. In vitro experiments revealed that the inner layer of the membranes facilitated attachment, proliferation, ingrowth, and osteogenic differentiation of hBMSCs. The three-layered membranes effectively supported new bone formation within a critical-size cranial defect in rats, rendering them highly efficient [178]. PLGA, known for its excellent biocompatibility, bone conductivity and low toxicity, is frequently utilized as a drug carrier and scaffold for BTE [179]. A multicomponent nano-nHAp-PLGA-Col membranes with mechanical properties similar to human cancellous bone prepared by melt pressing technique could promote MSCs adhesion, proliferation and osteogenic differentiation [180]. PVA, an FDA-approved water-soluble polymer, exhibits excellent biological characteristics such as hydrophilicity, biodegradability and biocompatibility. Previous studies have demonstrated that PVA can be combined with collagen to create various hybrid biocomposites including hydrogels and scaffolds for bone defect repair [181]. For example, fish collagen and PVA dual-layer membrane were prepared using a freezing/thawing and layer coating technique. Cell experiments demonstrated that this dual-layer membrane exhibited excellent cytocompatibility with rat BMSCs, while also significantly enhancing the expression of osteogenic genes (RUNX2, alkaline phosphatase (ALP), osteocalcin (OCN), and Col-I) in BMSCs [182].

8.2. Metal-based composites

Metals are extensively utilized in the treatment of medical diseases and injuries, such as tensile strength, stiffness, fracture toughness, and fatigue resistance. Specifically, titanium and titanium alloy implants exhibit robust bonding with bone tissue leading to excellent osseointegration [164]. Osseointegration is a crucial parameter for assessing the characteristics of bone repair materials. While metals exhibit favorable properties and are commonly employed in fracture fixation, they also pose several challenges, such as non-degradability. Moreover, their elastic modulus surpasses that of human cortical bone, leading to stress shielding. Stress shielding refers to the phenomenon where prolonged contact between the implant and the bone reduces mechanical loading on the bone, resulting in bone embrittlement or osteoporosis [183].

To achieve osseointegration, it is imperative to enhance osteoblastic differentiation and mitigate inflammatory cell responses. One effective approach to optimize this process involves surface modification of implants through coating techniques. Extensive studies have been conducted on collagen cross-linking for the purpose of stabilizing against enzymatic degeneration and ensuring prolonged structural and mechanical integrity during biomedical applications. The limited osseointegration capacity of 316 L stainless steel was successfully overcome by fabricating a composite coating using a spin coating technique, which consisted of polyelectrolyte multilayers comprising collagen, a chitosan barrier, and poly- γ -glutamic aci [184]. Veronesi et al. reported that titanium alloy (Ti-6Al-4V) coating with Col-I fabricated through electron beam melting, which improved osseointegration and bone growth of Ti-6Al-4V [185]. Müller et al. constructed tantalum oxide coating as a foundation for covalently immobilizing a collagen layer leads to further enhancement in the integration of implant tissue [186].

In recent years, there has been significant advancement in the development of diverse alloy materials for bone repair implants. Continuous research on the design and manufacturing of alloying materials has facilitated metal bone implants to effectively address the escalating clinical demand. Various alloy materials have undergone extensive investigation and optimization to better accommodate the repair and replacement of bone defects. However, due to their high elastic modulus, stress shielding effects are more pronounced. Additionally, a secondary surgical procedure is required for the removal of the implant. In order to address the limitations associated with conventional alloys in clinical applications, numerous researchers have been investigating and experimenting with collagen coating technology as a means to modify implant surfaces. Nevertheless, this approach is still in its research phase and poses various challenges, including manufacturing techniques and injection devices. With further advancements, these innovations are expected to be implemented in clinical settings for the development of metal-based bone implants.

8.3. Inorganic non-metallic materials-based composites

Inorganic non-metallic materials have high mechanical strength and are not easily deformed, such as bioceramics, which have been widely studied for the treatment of bone defects. Bioceramics including HA, β-TCP, calcium polyphosphate (CPP), and et al. as components are similar to the inherent inorganic components of natural bone, which have good biocompatibility, biodegradability, osteoconductivity and osteoinduction [187], that are extensively used in collagen scaffolds for bone regeneration. They not only enhance the mechanical strength and biological activity of pure collagen scaffolds, but also augment the osteoconductive capacity, dimensional stability, and surface area of the composite scaffold.

The incorporation of HA as the primary inorganic constituent of bone renders it an exceptional substitute material for bone regeneration, thereby enhancing the mechanical properties of composite materials. A 3D-printed biological scaffold composed of collagen, HA, and fibroin, incorporating recombinant human erythropoietin, for the reconstruction of mandibular defects [175]. Xing et al. developed a chitin/HA/collagen scaffold (CHCS) by employing epichlorohydrin as the crosslinking agent. The incorporation of HA not only augmented the compressive strength of CHCS but also facilitated the formation of calcium nodules owing to its osteoconductive properties. Histological staining demonstrated that collagen promoted collagen deposition and stimulated new bone formation. X-ray imaging further indicated that transplantation of CHCS expedited bone repair, underscoring its potential in facilitating bone regeneration [188]. β-TCP has attracted in bone grafting due to excellent osteoconductivity and resorbability. The composition and microstructure of this calcium phosphate ceramic are very close to those of natural bone, which is advantageous to provide a desirable environment for bone tissue regeneration [189]. The collagen/β-TCP scaffold was fabricated using the freeze-drying technique, which has been demonstrated to enhance vascularization and exhibit excellent integration with the surrounding tissue. Incorporating β-TCP powder into the porous collagen matrix effectively improved the mechanical and biological properties of the collagen scaffolds, making them promising bone substitutes for enhanced bone regeneration in orthopedic and dental applications [190]. Lee et al. showed a biomimetic composite composed of collagen and β-TCP as a BMP-2-delivering bone graft substitute to achieve a robust bone grafting and augmented bone regeneration [191].

However, different collagen-based biocomposites also possess distinct characteristics, encompassing advantages and limitations, as illustrated in Table 2. Currently, researchers are actively endeavoring to develop collagen scaffold materials with optimal properties for bone regeneration, including an interconnected porous structure, ppropriate pore size, exceptional mechanical strength, high biocompatibility and biodegradability levels, efficient bone conduction capabilities, as well as potent osteoinductive potential.

Table 2.

The advantages and limitations of collagen with composite components. PCL, polycaprolactone; PLGA, poly (lactic-co-glycolic acid; PVA, polyvinyl alcohol; HA, hydroxyapatite; β-TCP, β-Tricalcium Phosphate.

Materials	Examples	Advantages	limitations
Natural polymers	Alginate, chitosan, hyaluronan, silk fibroin	Abundant and easily accessible, good biodegradability and bioabsorbability, low immunogenicity, easy processing into injectable microbeads under mild physiological conditions	Poor cell adhesion, weak mechanical properties, rapid degradation
Synthetic polymers	PCL, PLGA, PVA	Good mechanical properties, ease of processing	Unsatisfactory cell adhesion, poor biological activity, slow biodegradability
Metals	Stainless steel, titanium alloy	Good tensile strength and stiffness	Non-degradability, stress shielding
Inorganic non-metallic materials	HA, β-TCP	Good biocompatibility, biodegradability, osteoconductivity and osteoinduction	Poor homogeneity, high brittleness

9. Conclusions and perspectives: new ideas and methods for the potential application of collagen

The genetically engineered protein polymers (GEPP) constitute a category of multifunctional materials distinguished by their precisely controlled molecular structure and property profile. This class of materials possesses several distinct advantages [[192], [193], [194]]. Firstly, GEPP allows for flexible combination of structural elements found in nature, such as three-layer helices, enabling precise control over both structural and material properties. Secondly, the protein sequence can incorporate the desired number of biological functional domains, including cell binding sites, facilitating the design of biomimetic scaffolds that closely resemble the native extracellular matrix and elicit specific cellular responses. Thirdly, GEPP exhibits minimal batch-to-batch variation due to its ability to consistently reproduce materials through a standardized production process involving fermentation and purification once a transgenic host organism has been constructed. Fourthly, this recombinant product and all its constituent building blocks are monodisperse as they are synthesized via a biosynthetic pathway that ensures their identical composition at an individual molecule level. Finally, concerns related to animal derivatives such as uncontrolled degradation or potential contamination with infectious agents have been effectively addressed in GEPP's approach. Han et al. developed collagen scaffolds engineered with modified collagen-binding BMP-2, demonstrating that the incorporation of a site-specific collagen binding domain enabled effective posterolateral intertransverse process fusion in rats at a remarkably low dose of 0.02 mg CBD-BMP-2/cm3 collagen scaffold. It suggested that combination delivery could serve as an alternative approach for spine fusion, mitigating the adverse effects associated with high doses of BMP-2 [195]. The application of nanotechnology in the field of regenerative medicine has witnessed a significant surge in recent decades. The exploration and development of materials capable of exerting precise control over biological processes at the nanoscale have garnered substantial attention, driven by our deepening comprehension of cellular interactions with biomaterials at this level [196]. Wang et al. conducted a study on the production and formation of nano collagen bone for alveolar ridge preservation, focusing on addressing poor post-tooth extraction alveolar ridge conditions resulting from delayed repair or inadequate dental/oral hygiene practices and knowledge. In order to protect the residual ridge after tooth extraction, artificial nano collagen bone was implanted in patients, followed by subsequent multi-slice computed tomography scans to evaluate the initial alveolar bone mineral density immediately after implantation. Three months later, a repeated scan revealed successful fusion between the residual ridge and the nano collagen artificial bone, accompanied by an observed increase in alveolar bone mineral density [197].

As genetic engineering and nanotechnology continue to improve, advances in different application areas can be expected. A combinatorial approach based on protein domain selection to generate novel collagen platforms allows fine-tuning of material properties for each specific application. In addition, different functions confer unique material properties that will provide alternative and better solutions for BTE applications and many other biomaterial interfaces. Overcoming synthetic related problems and creating a new generation of protein-based materials that mimic existing biopolymers, have signal transduction properties and match the mechanical properties of green production is now possible.

In the human body, collagen is the most abundant protein, accounting for about one-third of the body's protein tissue by weight. Collagen is a fibrous protein composed of the tissue's natural ECM. Collagen fibers and fibers are formed by the assembly and cross-linking of mature collagen molecules. They come in various forms from commercial sources. The research of collagen extracted from animal extracellular matrix is basically mature and has a wide range of applications in biomedicine, medicine and healthcare products. Bovine and porcine are classified as traditional sources of Col-I, and inevitably, each source has its own advantages and disadvantages, while scientists are eager to mimic the ECM environment, are pursuing biomimetic approaches to synthetic collagen. Since the ECM is a dynamic structure, the degradation of collagen in extracellular pathways involves certain members from the MMPs and cysteine protease. How to use the mechanism of different proteases on collagen degradation to regulate the process of bone regeneration that remains to be further studied. Fibrous collagen is abundant in nature and is used to obtain hydrogels, scaffolds, membranes, nanoparticles/particles, and for the transfer of bioactive agents. In the process of promoting bone regeneration, materials have been widely studied and applied in bone tissue construction. The incorporation of collagen into polymers, metals and so on, promotes bone regeneration. By simulating the structural or functional characteristics of natural collagen, we can understand the physicochemical factors responsible for functional ECM assembly, and overcome the biological defects such as the risk of infection, inflammatory response, and insufficient biological activity of collagen. However, at present, only a limited number of composites have received approval from the FDA for clinical use. These composites still encounter obstacles in terms of their mechanical properties, biological stability and activity, immune response, regional vascularization, and other safety concerns related to bone regeneration in humans. As a result, researchers are shifting their focus towards collagen-based materials that can be tailored to individual patients. By combining functional material manufacturing with the innate regenerative potential of patients instead of solely relying on materials themselves, new possibilities for clinical translation may emerge in the coming decade.

Disclosures

The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.

Declaration of competing interest

We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare.