Recombinant collagen in regenerative medicine: Expression strategies, structural design, and translational applications

Abstract

Recombinant collagen represents a new generation of biomaterials that integrate molecular precision, functional tunability, and scalable biomanufacturing. While animal-derived collagens remain clinically established, their inherent biological variability, limited controllability, and potential pathogen risks have spurred the development of recombinant systems capable of producing collagen with defined sequences and consistent quality. Advances in synthetic biology have enabled expression across diverse hosts—including E. coli, yeast, plants, mammalian cells, and transgenic organisms—each offering distinct advantages in yield, post-translational modification, and triple-helix assembly. Emerging molecular architectures, encompassing triple-helical recombinant collagens, non-helical gelatin-like proteins, and multifunctional fusion constructs, collectively expand the structural repertoire and functional landscape of recombinant collagen-based biomaterials. These engineered materials show strong promise in bone and cartilage regeneration, skin reconstruction, and corneal repair. Nonetheless, challenges remain in achieving complete hydroxylation, cost-effective large-scale manufacturing, and harmonized regulatory standards. The integration of AI-assisted sequence design, programmable molecular engineering, and GMP-compliant production is expected to accelerate clinical translation. By bridging molecular innovation with clinical application, recombinant collagen is poised to redefine the landscape of regenerative medicine and usher in a new era of precision-engineered biomaterials.

Graphical abstract

This review provides a comprehensive roadmap of recombinant collagen, spanning molecular design, biomanufacturing platforms and translational applications in regenerative medicine.

1. Introduction

Collagen is the fundamental structural and functional backbone of human connective tissues, constituting nearly one-third of total body protein and playing a central role in maintaining extracellular matrix (ECM) architecture, mediating cell–matrix interactions, and orchestrating tissue regeneration processes [[1], [2], [3]]. As the principal component of skin, bone, cartilage, and the vasculature, collagen provides essential mechanical support and biochemical cues, making it a cornerstone biomaterial for tissue engineering, wound healing, and regenerative medicine applications [[4], [5], [6], [7], [8], [9]]. Moreover, collagen-based three-dimensional (3D) bioprinting has enabled new paradigms in tissue fabrication and organ repair, offering spatially controlled construction of functional architectures [[10], [11], [12], [13]]. For decades, biomedical research and clinical practice have relied predominantly on collagen extracted from animal tissues [14]. However, animal-derived collagen presents several critical limitations, including potential immunogenicity, batch-to-batch variability, and the risk of zoonotic pathogen transmission, all of which hinder its use in advanced therapeutic settings. On the other hand, chemically synthesized collagen-mimetic peptides, while structurally well-defined, fail to recapitulate the molecular complexity and full bioactivity of native collagen due to restricted chain length, low production yields, and poor scalability [15,16]. These intrinsic shortcomings underscore the urgent need for alternative collagen sources that combine structural fidelity, biological functionality, and manufacturing feasibility.

The emergence of recombinant collagen represents a paradigm shift in collagen biotechnology. Enabled by advances in synthetic biology, protein engineering, and biomanufacturing, recombinant approaches allow for precise sequence design, controlled post-translational modification (PTM), and scalable production of molecules with biomimetic fidelity or enhanced functionalities [[17], [18], [19], [20]]. A variety of heterologous expression platforms — including bacteria, yeast, mammalian cells, plants, and transgenic animals — have been harnessed to produce structurally diverse collagen variants [21], spanning triple-helical constructs, gelatin-like polypeptides, and multifunctional fusion proteins. These innovations transcend many of the inherent limitations of animal-derived and chemically synthesized collagens, laying the foundation for next-generation biomaterials such as bioactive scaffolds, injectable hydrogels, and patient-specific regenerative therapies [22].

Despite these advances, a comprehensive framework that unifies molecular design principles, expression strategies, and translational applications of recombinant collagen remains lacking. Previous reviews have typically focused on individual host systems or isolated biomedical applications, leaving a fragmented understanding of this rapidly evolving field [20,21,23,24]. A holistic synthesis is therefore essential to bridge fundamental molecular insights with applied biomaterial development and to accelerate the clinical translation of recombinant collagen technologies.

Here, we present a critical and integrative review of recombinant collagen as a rising class of engineered biomaterials. We first survey current expression platforms and post-translational modification strategies that underpin efficient folding and stabilization of the collagen triple helix. We then classify the major structural formats — including triple-helical constructs, non-helical gelatin-like polypeptides, and multifunctional fusion proteins — and discuss their diverse applications in tissue regeneration, spanning bone, cartilage, skin, and corneal repair (Fig. 1). Furthermore, we examine key technical and translational bottlenecks, such as limited hydroxylation efficiency, suboptimal yield, and challenges in clinical-scale manufacturing, while highlighting promising advances in modular protein engineering, AI-assisted sequence design, and good manufacturing practice (GMP)-compliant production pipelines. By integrating molecular design principles with translational considerations, recombinant collagen is poised to redefine the landscape of regenerative medicine and usher in a new generation of high-performance, precision-engineered biomaterials.

Fig. 1.

Schematic overview of recombinant collagen biotechnology, illustrating expression platforms, structural engineering strategies, and representative applications in bone, skin, cornea and cartilage regeneration.

2. Structure, function, and preparation of collagen

2.1. Structural hierarchy

Collagen is defined by a repeating (Gly-Xaa-Yaa)_n motif and a distinctive right-handed triple-helical structure [25,26] (Fig. 2). Glycine (Gly), the smallest amino acid, appears at every third position, enabling the tight packing of three α-chains within the helix. The Xaa and Yaa positions are most frequently occupied by proline (Pro) and hydroxyproline (Hyp), residues that impose conformational constraints on the polypeptide backbone, stabilize the triple helix, and promote extensive intramolecular hydrogen bonding [[27], [28], [29]]. Three left-handed polyproline II–type helices, each offset by one residue, intertwine around a central axis to form a stable right-handed superhelix. This supramolecular structure is further stabilized by a dynamic hydration shell, where water molecules form transient interactions with polar side chains to strengthen the hydrogen-bonding network. These hydration-mediated interactions not only enhance the thermodynamic stability of the triple helix but also promote intermolecular association, thereby facilitating higher-order assembly [30,31].

Fig. 2.

Triple-helical structure of collagen and the hierarchical assembly of fibrillar collagen. (A) Triple helix formed by the repetitive sequences of (ProHypGly)₄–(ProHypAla)–(ProHypGly)₅. Reproduced with permission [26]. Copyright 2009, Annual Reviews. (B) Arrangement of the Pro X–Pro Y–Gly chain within the collagen triple helix. Reproduced with permission [29,37]. Copyright 2025, Oxford Univ Press. (C) Staggered alignment of the three polypeptide chains forming a ladder-like hydrogen-bonding network. (D) Extracellular assembly of collagen molecules into microfibrils. Reproduced with permission [36]. Copyright 2014, Nature Portfolio.

The self-assembly of collagen triple helices into fibrils with characteristic D-periodic banding is a tightly regulated and highly orchestrated process (Fig. 2). Procollagen precursors undergo enzymatic cleavage of their N- and C-terminal propeptides to yield mature collagen monomers, which align in a parallel, head-to-tail orientation along the longitudinal axis. Covalent crosslinking between adjacent molecules stabilizes the assembly into primary microfibrils, which adopt a quarter-staggered arrangement with an approximately 67 nm axial offset, producing the distinctive D-periodic banding pattern [32,33]. These microfibrils subsequently aggregate laterally to form fibrils, which further assemble into higher-order fibrous networks that impart tensile strength to connective tissues [25]. This hierarchical organization spans multiple length scales. In tendons, for example, type I collagen molecules first associate into elongated microfibrils (1–20 μm in diameter), which bundle into fascicles (150–1000 μm) and ultimately form tertiary fiber bundles (up to 3 mm). Such a multilevel structural hierarchy preserves tissue morphology and confers exceptional biomechanical reinforcement, enabling connective tissues to withstand substantial mechanical loads while maintaining structural integrity [[34], [35], [36]].

2.2. Biological functions

2.2.1. Structural scaffold

Collagen is the predominant structural protein of the extracellular matrix (ECM) and a fundamental determinant of tissue integrity and biomechanical stability [38]. It forms the primary scaffold of connective tissues — including bone, cartilage, and skin — where it provides tensile strength, structural organization, and a 3D framework that supports cellular architecture [39]. In bone, type I collagen assembles into highly ordered tubular fibers that serve as a template for mineral deposition and augment compressive strength [40,41]. In skin, collagen fibers adopt a random, mesh-like configuration, conferring elasticity and mechanical robustness against external stress [42]. In cartilage, type II collagen maintains matrix architecture and imparts resilience under compressive loading [43]. Type VII collagen contributes to dermal–epidermal cohesion by forming anchoring fibrils within the basement membrane, thereby supporting adhesion and barrier functions at the dermal–epidermal junction [44]. The evolutionary conservation of collagen's structural role is underscored by the presence of mammalian type I and III collagens within spongin — a biopolymer found in ancient poriferans — highlighting the primordial origin and enduring significance of collagen's mechanical functions [45]. Through coordinated interactions among diverse collagen subtypes, the ECM establishes a robust, hierarchical scaffold that preserves tissue morphology and sustains the mechanical integrity of multicellular organisms.

2.2.2. Cell regulation

Collagen is not merely a structural scaffold but also an active regulator of cellular behavior, mediating a wide range of biological processes — including cell adhesion, signal transduction, and matrix remodeling — primarily through interactions with cell surface receptors [46]. Among these, integrins are the principal mediators of collagen–cell interactions. They recognize the canonical GFOGER motif within the collagen triple helix, thereby regulating adhesion, migration, and downstream signaling events [47]. Specific integrin heterodimers exhibit distinct collagen-binding preferences: α1β1 binds to types I, III, IV, IX, XIII, and XVI [48,49]; α2β1 engages types I, IV, VI, and XII [50]; and α11β1 interacts with types I, IV, V, and IX [51].

Discoidin domain receptors (DDRs), a family of receptor tyrosine kinases that detect fibrillar collagens, bind conserved hexapeptide motifs and initiate signaling cascades that regulate tissue homeostasis and remodeling. DDR1 preferentially recognizes type IV collagen [52], whereas DDR2 binds GVMGFO motifs, leading to the upregulation of matrix metalloproteinases (MMPs) and promoting matrix remodeling [53]. Additionally, glycoprotein VI (GPVI), a collagen receptor uniquely expressed on platelets and megakaryocytes, preferentially binds to type I and III collagens, thereby triggering platelet activation and promoting thrombus formation [54]. The osteoclast-associated receptor (OSCAR), an activating receptor found on osteoclasts and macrophages, recognizes GPOGPAGFO repeat motifs within types I, II, and III collagens, thereby modulating osteoclast differentiation and contributing to immune regulation [54]. Through these diverse receptor-mediated pathways, collagen orchestrates complex cellular responses that extend far beyond its structural role, positioning it as a central regulator of tissue development, remodeling, and homeostasis.

2.2.3. Tissue regeneration and remodeling

Collagen plays a pivotal role in tissue regeneration and remodeling by modulating cytokine activity and intracellular signaling pathways. In skin wound healing, collagen interacts directly with platelets to trigger activation and initiate primary hemostasis, forming a provisional matrix that not only prevents pathogen invasion but also recruits inflammatory cells to the injury site [55,56]. It further acts synergistically with growth factors such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), as well as chemokines like CXCL12, to regulate the local microenvironment, promote neovascularization, and facilitate keratinocyte migration, thereby accelerating re-epithelialization [55,56].

In bone tissue remodeling, collagen activates key signaling pathways, including extracellular signal-regulated kinase (ERK), protein kinase B (PKB/Akt), and mitogen-activated protein kinase (MAPK), to regulate the expression of various osteogenesis-related genes, such as alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), and type I collagen (Col-I) [[57], [58], [59]]. Simultaneously, collagen modulates osteoclast differentiation by regulating the expression of receptor activator of nuclear factor κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), and cathepsin K, thereby maintaining a finely tuned balance between bone formation and resorption [60]. Through these multifaceted functions, collagen orchestrates coordinated cellular and molecular events essential for tissue repair and structural remodeling, underscoring its dual role as both a physical framework and a dynamic signaling hub in regenerative processes.

2.3. Preparation strategies

2.3.1. Extraction from animal tissues

Animal tissues have long served as the primary source of collagen, with both terrestrial and marine organisms providing abundant raw materials. Among terrestrial sources, bovine tissues — commonly obtained from dairy cattle, yaks, and buffalo — are the most widely utilized [61]. These tissues contain a diverse array of collagen types: type I predominates in skin, bone, and tendons; type II is abundant in nasal and articular cartilage; type III is enriched in the placenta and neonatal calf skin; and type IV is found in the cornea and placenta [62]. Despite their versatility, bovine-derived collagens pose potential biosafety concerns, including the risk of transmitting zoonotic diseases such as bovine spongiform encephalopathy and foot-and-mouth disease. Moreover, interindividual variations in age, breed, and environmental conditions can affect collagen purity, thermal stability, and biological activity, leading to substantial batch-to-batch variability [63]. Porcine tissues, particularly skin and tendons, represent another major terrestrial source of type I collagen, while cartilage is rich in type II collagen; however, their use is limited by religious restrictions and biosafety concerns [64].

Marine organisms such as carp and mackerel have emerged as attractive alternative sources of collagen [65]. Compared with terrestrial collagens, marine-derived collagens exhibit several notable advantages, including lower immunogenicity, superior biocompatibility, and a markedly reduced risk of mammalian pathogen transmission [66,67]. Nevertheless, their relatively low proline and hydroxyproline content results in reduced thermal stability and complicates the extraction process. In summary, while animal-derived collagen remains a readily available and compositionally diverse source of biomaterial, issues such as structural degradation during extraction, significant batch variability, and unresolved biosafety concerns continue to limit its application in advanced biomedical materials and regenerative therapies.

2.3.2. Chemical synthesis of collagen-like peptides

Chemical synthesis represents a powerful strategy for constructing collagen-mimetic molecules, particularly suited for generating short peptide fragments that emulate the structural features and biological functions of native collagen [68]. Among various synthetic methods, solid-phase peptide synthesis (SPPS) is the most widely employed, enabling the efficient assembly of mini-collagens containing repetitive Gly-Xaa-Yaa motifs. This approach facilitates the formation of stable triple-helical structures and even allows the rational design of heterotrimeric assemblies [69,70].

Several landmark studies have demonstrated the potential of chemically synthesized collagen mimetics. Kakiuchi et al. synthesized homotrimeric mini-collagens (PPG)_n (n = 10 or 20) exhibiting well-defined triple-helical architecture [71]. Horng et al. employed (POG)₈ as a model system to investigate the impact of glycine substitutions on helix stability [72,73]. Hartgerink et al. constructed heterotrimers composed of (DOG)₁₀, (PKG)₁₀, and (POG)₁₀, achieving a thermal denaturation temperature of 65 °C [74]. Hu et al. further incorporated adhesive modules into synthetic sequences to promote fibrous self-assembly, successfully recapitulating the banded supramolecular architecture characteristic of native collagen [75]. Moreover, embedding bioactive motifs such as GFOGER has been shown to restore integrin-binding functionality that is otherwise lost in fragmented collagen [76,77]. Despite enabling precise structural control, chemical synthesis remains limited by short achievable chain lengths, high production costs, low yields, and potential cytotoxicity associated with residual solvents. These constraints severely restrict its utility to structural and mechanistic studies or as functional motifs in biomaterial design, while scalability and clinical translation continue to present major challenges.

2.3.3. Recombinant expression systems

Recombinant technology has emerged as a pivotal strategy for collagen production, offering precise molecular control and the ability to engineer tailored functionality. By introducing optimized collagen genes into heterologous host systems and inducing expression under defined conditions, recombinant approaches enable the biosynthesis of collagen with high structural fidelity and programmable bioactivity [37,78,79]. A variety of expression platforms have been successfully developed: Escherichia coli facilitates the expression of tyrosine-rich biomimetic collagens [80]; Pichia pastoris enables the secretion of human type III collagen adopting a triple-helical conformation [81]; mammalian cells, such as human dermal fibroblasts and CHO cells, produce type I and III procollagens as well as type IV complexes [82,83]; and plant-based systems, exemplified by transgenic tobacco, have also been engineered to yield structurally stable type I collagen [84]. Collectively, these expression systems allow the production of multiple collagen subtypes with precise sequence control, defined post-translational modifications(PTMs), and tunable biological functions, positioning recombinant technology as a robust and versatile alternative to native collagen sources.

To conclude, collagen production currently relies on three principal strategies — animal tissue extraction, chemical synthesis, and recombinant expression — each with distinct strengths and inherent limitations (Table 1). Extraction from animal tissues is well established and benefits from abundant raw materials, yet batch-to-batch variability, immunogenicity, and pathogen transmission risk restrict its use in advanced biomedical contexts. Chemical synthesis offers unparalleled precision in molecular design, making it invaluable for mechanistic studies and functional peptide development; however, short chain lengths, high costs, and low yields hinder its scalability and clinical translation. Recombinant technology combines high biosafety with molecular programmability and is increasingly becoming the dominant approach for collagen research and biomedical applications. Nonetheless, significant challenges remain, including improving production yield, achieving complete and consistent PTMs, and reducing the costs associated with large-scale manufacturing.

Table 1.

Comparison of major strategies for collagen production.

Method	Tissue Extraction	Chemical Synthesis	Recombinant Expression
Source	Animal skin, bone, tendon, and cartilage (bovine, porcine, marine)	Synthetic short-chain collagen-mimetic peptides	E. coli, yeast, mammalian cells, transgenic plants
Advantages	Well-established process; abundant raw materials; multiple collagen subtypes	High molecular precision; customizable sequences and bioactive motifs	High biosafety; precise sequence and functional programmability
Limitations	Immunogenicity; pathogen transmission risk; batch-to-batch variability	Short chain length; high cost and low yield; potential cytotoxicity from residual solvents	Incomplete PTM; limited self-assembly capacity; scalability and cost challenges
Applications	Conventional medical and cosmetic materials	Structural and mechanistic studies; functional peptide development	Emerging biomedical and industrial applications

3. Expression systems of recombinant collagen

3.1. Prokaryotic expression system

3.1.1. Escherichia coli

Escherichia coli (E. coli) is one of the most widely used prokaryotic hosts for recombinant protein production due to its low cost, rapid growth, ease of genetic manipulation, well-characterized genome, and high capacity for heterologous gene expression [85,86]. It has been extensively employed to produce recombinant collagens containing repetitive sequences, triple-helix motifs, or post-translational hydroxylation modifications. Early efforts focused on expressing artificially constructed repetitive (Gly-Xaa-Yaa) tripeptide sequences in E. coli. Goldberg et al. expressed type II collagen with 32 Gly-Pro-Pro repeats under a λpL promoter, although the protein accumulated as inclusion bodies [87]. Ma et al. produced tandem fragments of the human type I collagen α1 chain [88], while Yao et al. generated functional collagen-derived peptides [89]. However, repetitive sequences frequently suffer from genetic instability, which reduces both yield and structural integrity [90].

Several bacterially derived collagen-like proteins have been expressed in E. coli. Vandersmissen et al. reported the expression of the Legionella pneumophila collagen-like protein Lcl [91], while Boydston et al. confirmed that BclA collagen forms a triple helix with a melting temperature of 37 °C, comparable to that of mammalian collagen [92]. The Scl1 and Scl2 proteins from Streptococcus pyogenes exhibit canonical collagen triple-helical structures, promote L929 fibroblast proliferation, and elicit no detectable immune response in murine models [93,94]. Although these proteins lack proline hydroxylation, they nonetheless maintain a stable triple-helical structure.

A major limitation of E. coli is the absence of prolyl 4-hydroxylase (P4H) and lysyl hydroxylase (LH), enzymes essential for post-translational modification(PTM), which constrains its ability to produce biologically functional collagen. To address this challenge, exogenous hydroxylases have been introduced into the host. Buechter et al. expressed prolyl-tRNA hydroxylase under hyperosmotic conditions with hydroxyproline supplementation, resulting in partially hydroxylated triple helices; however, hydroxyproline was incorporated nonspecifically into both the Xaa and Yaa positions, compromising site specificity and potentially affecting structural and functional uniformity [95]. Rutschmann et al. co-expressed human type III collagen with mimivirus-derived P4H and LH, achieving hydroxylation levels of approximately 25 % for proline and lysine residues, thereby enhancing helix stability [96].

In summary, the E. coli expression system remains a cornerstone for the industrial production of recombinant collagen due to its cost-effectiveness, ease of genetic manipulation, high expression efficiency, and compatibility with well-established fermentation platforms [23]. Strategies such as media optimization, vector engineering, promoter tuning, fusion tag selection, chaperone co-expression, and soluble expression techniques have significantly improved protein yield and structural quality [97,98]. Moreover, E. coli's robust and flexible genetic toolkit enables complex metabolic engineering and precise regulatory control, laying a strong foundation for low-cost, scalable, and tunable collagen biosynthesis. Nevertheless, the absence of endogenous PTMs machinery continues to represent a major barrier to the production of fully hydroxylated and structurally complete triple-helical collagen.

3.1.2. Bacillus species

Bacillus species represent an alternative prokaryotic expression system, offering key safety advantages due to their lack of endotoxins, strong secretion capacity, and well-established industrial use in enzyme and pharmaceutical manufacturing [99,100]. Bacillus pumilus has been employed to express gelatin-like proteins through fused peptide constructs; however, its scalability is limited by low transformation efficiency, stringent culture requirements, and the absence of standardized production protocols [101]. In contrast, Bacillus subtilis provides a more mature expression framework, yet challenges such as plasmid instability and proteolytic degradation by host-secreted proteases persist. The use of protease-deficient mutants or protease inhibitors can mitigate these issues but often adds cost and complexity to the production process [102,103]. Consequently, while Bacillus hosts show considerable promise for the secretion of small collagen fragments or gelatin derivatives, substantial strain engineering and process optimization are required to achieve efficient production of structurally complete and biologically active collagens.

3.2. Eukaryotic expression system

3.2.1. Yeast

Yeasts combine rapid growth kinetics, eukaryotic PTM capabilities, and efficient secretion pathways, making them attractive hosts for recombinant protein production [104,105]. The principal yeast species explored for collagen biosynthesis include Pichia pastoris (P. pastoris), Saccharomyces cerevisiae (S. cerevisiae), and Hansenula polymorpha (H. polymorpha).

• (1)Pichia pastoris

As the most widely employed yeast host, P. pastoris offers strong promoter-driven expression, efficient secretion, and compatibility with high-density fermentation [106,107]. Myllyharju et al. co-expressed the full-length human type I collagen α1(I) chain with P4H, obtaining correctly folded triple-helical structures [108]. Vuorela et al. utilized an α/αMF–PDI/proα1(III) expression system to co-express human type III collagen proα1(III) with P4H, yielding triple-helical collagen with 51.6 % hydroxylation and a melting temperature of ∼39 °C [81]. Fang et al. achieved co-expression of the human type III collagen α1 chain in the GS115 strain [109]. Despite these advances, Myers et al. reported that large procollagen molecules frequently accumulate in the endoplasmic reticulum (ER), thereby limiting secretion efficiency, while insufficient lysine hydroxylation and the absence of glycosylation further compromise triple-helix stability and biological functionality [110].

To overcome these secretion bottlenecks, multi-dimensional optimization strategies have been developed at both molecular and process levels. At the molecular level, modifying the α-factor signal peptide, incorporating human serum albumin (HSA) fragments, or fusing small chaperone peptides can enhance precursor translocation and folding, thereby reducing misfolding and aggregation [[111], [112], [113]]. At the process level, fine-tuning fermentation conditions — including dissolved oxygen concentration, carbon source ratios, and induction temperature — is critical. Lowering the induction temperature to 20–25 °C and optimizing methanol/glycerol feeding profiles can more than double recombinant yields, while cofactors such as ascorbate and iron enhance P4H activity and triple-helix stability [114]. Wang et al. reported a yield of 10.3 g/L recombinant human type III collagen in a 5 L fermenter through gene copy number optimization and chaperone co-expression [115]. Additional advances in promoter engineering and feeding strategies have further improved both yield and molecular stability [116,117]. Collectively, P. pastoris has emerged as a leading industrial expression system for recombinant collagen, though further refinements are necessary to achieve precise glycosylation control.

• (2)Saccharomyces cerevisiae

S. cerevisiae is a robust and versatile eukaryotic host capable of both intracellular expression and extracellular secretion of recombinant proteins via signal peptide–mediated pathways [[118], [119], [120], [121]]. Vaughan et al. expressed recombinant human type III collagen with incorporated hydroxyproline residues [122], and Toman et al. produced type I procollagen heterotrimers with hydroxylation levels (∼82 %) approaching those of native collagen [123]. Using the α1β1-MCOL and α2β2-MCOL systems, type III collagen trimers were assembled with melting temperatures only 3–5 °C below native collagen [124], and full-length human type III collagen was also successfully expressed, albeit at relatively low yields [125]. Despite these advances, the incomplete PTM machinery of S. cerevisiae — particularly its limited hydroxylation capacity — continues to restrict the efficiency and stability of collagen production.

• (3)Hansenula polymorpha

H. polymorpha exhibits significant potential for industrial-scale fermentation and the production of complex heterologous proteins due to its robust inducible promoters, thermotolerance, and efficient multi-copy gene integration [126]. However, its application to recombinant collagen synthesis remains in the exploratory stage. De Bruin et al. reported partial hydroxylation in gelatin-like proteins expressed in this system, even without co-expression of heterologous P4H [127]. Subsequent genomic analyses, however, confirmed the absence of endogenous P4H genes, suggesting that these earlier observations may have been detection artifacts [128]. To date, systematic co-expression with exogenous P4H has not been explored, leaving the system's full potential largely untapped.

3.2.2. Mammalian cell

Mammalian hosts provide a complete PTM machinery, including P4H, LH, and glycosylation enzymes, which together ensure efficient hydroxylation, glycosylation, and proper triple-helix folding [129]. This endows mammalian systems with a distinct advantage in producing functional recombinant collagens that closely replicate the structural fidelity and bioactivity of native molecules. A wide range of mammalian cell lines have been employed for stable collagen expression, including MOV-13, NIH 3T3, CHO, HT1080, COS, and HEK293 cells.

Chinese hamster ovary (CHO) cells have been widely used to express stable type IV and type VII collagens [83,130,131]. HT1080 cells secrete type I and II collagens at milligram yields [132,133], while HEK293 cells support specialized constructs, such as α1(X) collagen with enhanced hydroxylation [134,135]. Fichard et al. reported that α1(V) collagen expressed in the 293-EBNA system forms a stable triple helix with a denaturation temperature of 37.5 °C [136]. Additionally, NIH 3T3 and MOV-13 mouse fibroblasts have been employed for the expression of human procollagen and type I collagen [137,138]. Overall, mammalian cell systems yield recombinant collagens that most closely resemble native conformations, establishing a critical platform for high-end functional collagen research and therapeutic development. However, their broader application remains limited by relatively low production yields and high cultivation costs, which pose significant challenges for large-scale manufacturing and clinical translation.

3.2.3. Plant expression systems

Plant-based expression systems offer several inherent advantages — including high biosafety, scalability and minimal risk of contamination — making them attractive platforms for the production of medical- and nutrition-grade recombinant collagens [139,140]. Although plant cells naturally possess P4H-like enzymes, their intrinsic hydroxylation capacity is limited [139,141]. As a result, plant-derived recombinant collagens are typically non-hydroxylated or exhibit only minimal hydroxylation. For example, Zhang et al. expressed a fragment of human type I collagen in maize seeds and detected only trace levels of proline hydroxylation and no glycosylation [142]. Consistently, other studies have reported hydroxyproline contents below 2 % in plant-derived collagens [143]. Similar deficiencies were observed in barley cell cultures and seed-based systems, which failed to support adequate hydroxylation or other essential PTMs [144,145]. Ruggiero et al. further demonstrated that although homotrimeric human collagen expressed in tobacco could be proteolytically processed into mature forms, complete hydroxylation was not achieved [139].

To address these limitations, co-expression strategies involving human post-translational modification enzymes have been widely explored. Stein et al. co-expressed heterotrimeric type I collagen with P4H and lysyl hydroxylase 3 (LH3) in tobacco, producing a stable triple helix closely resembling native human collagen [146]. Xu et al. achieved hydroxyproline levels of 18.11 % in maize by co-expressing the α1(I) chain with P4H, approaching native levels [147]. Likewise, Shoseyov et al. co-expressed type I collagen with P4H and LH3 in tobacco vacuoles, yielding proteins with enhanced thermal stability and biological activity [148]. This strategy was further refined using a transient tobacco expression system incorporating a chimeric P4H composed of the Caenorhabditis elegans α-subunit and the mouse β-subunit, which significantly improved hydroxylation efficiency [141]. Overall, plant-based systems hold significant promise as scalable and safe platforms for recombinant collagen production. However, the faithful recapitulation of native PTMs remains a major challenge, representing a key barrier to achieving fully functional collagen suitable for clinical translation.

3.2.4. Transgenic animal expression systems

Transgenic animal systems offer unique advantages for recombinant collagen production owing to their comprehensive PTMmachinery and intrinsic capacity to correctly fold structurally complex proteins. These features enable the generation of collagen molecules with native-like modifications and functional properties. Toman et al. generated transgenic mice in which expression of a homotrimeric human type I procollagen, driven by the αS1-casein mammary gland–specific promoter, yielded a soluble triple-helical form, [(α1)₃]I, in milk despite limited hydroxylation [78]. Adachi et al. employed the posterior silk gland of silkworms to co-express human type III collagen with the α-subunit of P4H, yielding fibers containing hydroxyproline residues [149]. By introducing the baculovirus early transcriptional activator gene IE1, a homozygous transgenic silkworm strain was established, increasing the expression level of human type I collagen in cocoons from 0.8 % to 8.0 %. Despite this substantial yield improvement, the resulting collagen lacked a canonical triple-helical structure [150]. Similarly, Qi et al. expressed recombinant human type II collagen in silkworm epidermal tissue, achieving yields of approximately 1 mg per individual [151].

While transgenic animal systems are capable of introducing native-like PTMs and producing functional collagen molecules, they face significant challenges, including low expression efficiency, limited scalability, and additional ethical and biosafety concerns. As such, their application remains primarily confined to experimental studies, and considerable technological advancements will be required before they can support large-scale, clinically relevant collagen production.

3.3. Selection of expression systems

The host systems available for recombinant collagen production encompass E. coli, Bacillus species, yeasts, plants, insect and mammalian cells, as well as transgenic animals. Each system offers distinct advantages and inherent limitations in terms of yield, PTMs capacity, production cost, and industrial feasibility (Table 2). E. coli remains the most widely used prokaryotic host owing to its ease of genetic manipulation, rapid growth, low cultivation cost, and high expression efficiency. It is particularly suited for the rapid production of collagen fragments used in structural, functional, and mechanistic studies. However, the absence of essential PTMs — such as proline and lysine hydroxylation and glycosylation — together with the risk of endotoxin contamination, poses a major challenge to the production of fully functional, triple-helical collagen. By comparison, Bacillus species offer superior extracellular secretion and biosafety advantages but are hindered by lower yields, immature expression toolkits, and reduced genetic stability.

Table 2.

Comparison of expression systems for recombinant collagen.

Expression system	Typical hosts	Post-translational modification	Yield	Representative collagen constructs	Advantages	Limitations	Applications
Prokaryotic	E. coli, Bacillus	Absent (no hydroxylation or glycosylation)	High (mg–g L−1; up to 6–10 g L−1 in high-density fermentation)	Collagen fragments; bacterial collagens; human-like collagens; procollagen trimers (human type I α1, α2); engineered gelatin	Genetically tractable; cost-effective; robust fermentation; high yield	Lacks essential PTMs; incomplete triple-helix formation; endotoxin risk	Mechanistic studies; protein engineering; collagen fragment production
Yeast	S. cerevisiae, P. pastoris, H. polymorpha	Incomplete proline hydroxylation; limited glycosylation	High (g L−1)	Human type I, III collagen; gelatin; Procollagen trimers (human I-α1, 2); Human-like collagen	GRAS-certified; high secretion and yield; scalable fermentation; industrially proven	Partial PTMs; inappropriate glycosylation; methanol induction required	Functional recombinant collagen production; preclinical applications
Plant	Tobacco, barley, maize	Limited hydroxylation	Medium (0.5–50 mg kg−1)	Collagen (human I-α1, 2); Collagen fragment (human I-α1)	High biosafety; structural similarity to native collagen	Prolonged cycle, poor hydroxylation, and heterogeneity	Nutritional and biomedical applications; early translational research
Insect	Baculovirus-insect cells; silkworm	Relatively complete PTMs	Medium (0.5–60 mg L−1 in cells; ∼1 mg per larva)	Collagen I, II, III, IX resembling native collagen; Collagen (human IX-α1, 2,3)	Native-like PTMs; strong secretion capacity (e.g., silk glands)	Limited yield; batch variability; process complexity	Specialized biomedical materials; functional collagen production
Mammalian cells	CHO, HEK293, HT1080, NIH 3T3	Complete PTMs	Low–medium (0.01–0.6 g L−1)	Human procollagen I, II, VI, VII; collagen IV fragments	Native-like structure and functionality; accurate triple-helix formation	Extremely high cost; low scalability	High-end medical collagen development; therapeutic protein production
Transgenic animal	Mouse, silkworm	Complete PTMs	0.8–8 % of total protein (milk or silk)	Procollagen (human I-α2, III-α1)	Native-like modifications; high biological relevance	Low expression efficiency; limited scalability; ethical and biosafety concerns	Exploratory platforms; biomedical research

Yeast systems provide a balance between yield and partial PTM capabilities. Both S. cerevisiae and P. pastoris are recognized as GRAS (Generally Recognized As Safe) organisms by the U.S. Food and Drug Administration (FDA). S. cerevisiae benefits from extensive industrial use and well-established molecular tools, although excessive glycosylation can lead to immunogenicity. P. pastoris, by contrast, is distinguished by its high secretion efficiency, low glycosylation levels, and gram-per-liter yields, supported by robust large-scale fermentation platforms. The recent development of methanol-free induction systems further enhances its industrial applicability [152,153].

Plant-based expression systems offer significant advantages in terms of scalable and potentially cost-effective production, as well as the ability to generate collagen-like proteins that closely resemble native structures. Nevertheless, their translation to clinical and industrial use remains limited by long cultivation cycles, insufficient hydroxylation capacity, slow research progress, and biosafety concerns related to transgene containment. Mammalian cells provide the most accurate PTMs and yield recombinant collagens that most closely mimic native proteins, but their application is constrained by low productivity and high cost, limiting scalability. Insect cell systems can maintain biological activity and are compatible with complex folding requirements, yet they face similar challenges in yield and cost.

Transgenic animals, such as mice and silkworms, enable tissue-specific secretion of recombinant collagen — for example, in mammary glands or silk glands — offering unique advantages for producing functional proteins with native-like modifications. However, their use is constrained by low expression efficiency, stability challenges, and ethical considerations. Beyond conventional hosts, several emerging systems are gaining traction. Kluyveromyces marxianus, with its exceptionally high protein secretion capacity, is considered a promising future industrial platform [154]. Yarrowia lipolytica exhibits robustness under extreme conditions, offering advantages for specialized bioprocesses [155]. Lactococcus lactis, a widely used food-grade probiotic, shows potential for safe and functional collagen production, broadening its utility beyond food applications [156].

From an economic perspective, production costs can be broadly ranked as follows: mammalian systems > transgenic animals > insect systems > plant systems > yeast systems > prokaryotic systems. In current practice, E. coli remains the leading platform for laboratory research and scalable pilot production, serving as a workhorse for the rapid expression of collagen fragments in structural and functional studies. In contrast, P. pastoris has emerged as one of the most promising platforms for industrial-scale production, striking an optimal balance between yield, PTMs fidelity, and commercial feasibility. Looking ahead, the integration of advanced biofoundry infrastructure, automated high-throughput screening technologies, and AI-assisted sequence design is expected to further shorten development cycles, enhance product quality and batch-to-batch consistency, and reduce manufacturing costs — thereby accelerating the industrial deployment and clinical translation of recombinant collagen.

4. Classification and molecular design of recombinant collagen

Recombinant collagen can be broadly classified into three categories based on structural and functional attributes: triple-helical, non-triple-helical, and fusion types. Triple-helical collagens preserve the canonical native conformation, providing superior thermal stability, mechanical strength, and biocompatibility—properties indispensable for tissue engineering and regenerative medicine, where structural fidelity is paramount. Non-triple-helical variants, typically composed of short peptide fragments or gelatin derivatives, lack a complete helix yet retain key physicochemical characteristics. Their high solubility and facile processability make them particularly valuable for food, cosmetic, and certain biomedical applications. Fusion-type collagens integrate bioactive proteins or functional motifs with the collagen backbone, yielding multifunctional biomaterials with enhanced biological activity and broadened therapeutic potential. Each type offers unique advantages in design, function, and application, underscoring their significant promise for both scientific innovation and industrial translation.

4.1. Triple helical recombinant collagen

4.1.1. Strategies for producing stable triple helices

The triple-helical conformation is the defining structural hallmark of properly folded, biologically functional collagen and is widely regarded as the “gold standard” for structural validation. Strategies to stabilize recombinant collagen in this conformation generally fall into three main categories: direct expression of collagen backbone sequences, incorporation of terminal stabilizing domains to facilitate proper folding and prevent misassembly, and co-expression of prolyl 4-hydroxylase (P4H) to enhance post-translational modification and helix stability (Fig. 3).

Fig. 3.

Strategies for engineering triple-helical recombinant collagen. (A) Direct expression of the collagen backbone sequences with stable triple-helical structure; (B) Introduction of stabilizing peptides at the N- and/or C-termini of the collagen domain; (C) Co-expression with prolyl 4-hydroxylase (P4H).

Boydston et al. engineered a recombinant construct composed of an N-terminal V-domain, a central collagenous backbone, and a C-terminal structural motif, which exhibited a stable triple helix with high thermal stability [92]. Similarly, Xu et al. designed a construct containing a globular V-domain, 78 repeats of the (Gly-Xaa-Yaa) motif, and a C-terminal anchoring region, which also folded into a stable triple helix [157]. Bacterial-derived collagen-like proteins retaining the Gly-Xaa-Yaa motif have demonstrated spontaneous assembly into stable helices even in the absence of hydroxyproline, with melting temperatures of 35–39 °C, comparable to mammalian counterparts [[158], [159], [160]].

Incorporating stabilizing peptide motifs at the N- and/or C-termini is a key strategy for reinforcing triple-helical structures, as these domains guide proper assembly and protect against misfolding or degradation. Kaur et al. developed Col108, composed of quasi-repetitive collagen modules interspersed with (Gly-Pro-Pro)₄ sequences and flanked by Gly-Pro-Cys-Cys motifs, enabling disulfide crosslinking and enhanced stability [161]. Coiled-coil domains [162] and the foldon domain from bacteriophage T4 [163] have also been fused to promote helix nucleation and fibril assembly. Bai et al. further improved thermal stability by coupling C-terminal poly (Gly-Pro-Pro)_n sequences with disulfide crosslinking between foldon and the collagen backbone [164].

Co-expression of P4H represents a pivotal strategy for producing structurally stable recombinant collagens. Rutschmann et al. introduced virus-derived P4H/LH, attaining human type III collagen fragments with elevated hydroxylation levels [165]. Toman et al. achieved stable synthesis of type I and type III collagens via co-expression of human or chicken P4H [123,124]. Fang et al. similarly generated hydroxylated type III collagen by co-expressing viral P4H [109], while Wang et al. co-expressed type III collagen variants with bacterial P4H, yielding up to 2.54 g L⁻¹ of hydroxylated protein with a stable triple-helical structure [166].

Hyp is a critical post-translational modification that underpins collagen's triple-helix stability, mechanical strength, and resistance to proteolytic degradation. Although partially hydroxylated collagens can still support fibroblast adhesion and migration, insufficient hydroxylation may severely impair self-assembly, fibrillogenesis, and long-term implant performance, while potentially exposing immunogenic epitopes [167,168]. Conventional in vivo P4H co-expression can partially restore hydroxylation but is often constrained by low yield, uneven modification efficiency, and host incompatibility. To overcome these limitations, in vitro chemical and enzymatic strategies have been explored. Buechter et al. enhanced hydroxylation of human type I collagen α1 fragments by combining exogenous Hyp supplementation with prolyl-tRNA hydroxylase induction [95]. Cheng et al. employed proline-auxotrophic E. coli with precisely controlled Pro/Hyp feeding ratios to achieve tunable hydroxylation levels of 0–88 %, significantly improving thermal stability and cell-binding capacity [169]. Emerging approaches such as engineered P4H variants with broadened substrate specificity, multi-enzyme cooperative systems, and microfluidics-based continuous reaction platforms are further expanding the potential for scalable, automated hydroxylation [170].

Future optimization strategies are likely to focus on directed evolution of P4H to enhance catalytic efficiency and substrate scope, regulation of cofactor availability (e.g., α-ketoglutarate, ascorbate, and Fe²⁺) to boost enzymatic activity, and genome-scale metabolic engineering (e.g., CRISPR-based network remodeling) to create more favorable intracellular modification environments. Increasingly, hybrid approaches that integrate partial in vivo hydroxylation with precise in vitro modification are emerging as powerful solutions that balance efficiency and precision. Coupled with AI-guided enzyme engineering, these strategies hold strong potential for the scalable production of high-quality, structurally stable recombinant collagens and for accelerating their clinical translation.

4.1.2. Mechanisms of fibrillar assembly

Collagen fibrils, defined by their characteristic D-periodic banding pattern, represent the hallmark of hierarchical extracellular matrix architecture and are central to collagen's exceptional biomechanical resilience. Although recombinant collagens can reliably form triple helices, their ability to fully recapitulate native fibrillogenesis remains limited. For example, Perret et al. reported that transgenic plant-derived type I collagen successfully folded into triple helices but failed to assemble into canonical banded fibrils under physiological ionic strength [171]. Yoshizumi et al. engineered a collagen-like protein that aggregated into 4–5 nm subunits under neutral pH and high concentrations, which subsequently coiled into fibrillar structures; however, these assemblies lacked the regular D-periodicity and hierarchical order characteristic of native collagen [172]. Collectively, these studies indicate that while recombinant collagens possess inherent fibrillogenic potential, their supramolecular assemblies often fall short of native structural precision and mechanical robustness.

Compared with native collagen, recombinant variants typically exhibit three key deficiencies: (i) low self-assembly efficiency, often requiring non-physiological conditions to initiate fibrillogenesis; (ii) absence of canonical D-periodicity and well-defined hierarchical organization; and (iii) reduced thermal and mechanical stability. These shortcomings largely arise from insufficient hydroxylation, increased molecular flexibility, and altered charge distribution or hydration dynamics, which collectively disrupt molecular alignment and crosslinking during assembly.

Despite these limitations, several promising strategies are emerging to bridge the gap between recombinant and native fibrillar organization. Enhancing hydroxyproline content improves molecular rigidity and interchain interactions, while the incorporation of self-assembly-promoting motifs can facilitate nucleation and lateral association. Additionally, AI-guided sequence design offers powerful opportunities to engineer fibrillogenic domains with optimized charge distribution, hydrophobicity, and binding interfaces to drive controlled assembly. Achieving near-native hierarchical architecture—while preserving molecular uniformity, biosafety, and manufacturing scalability—would significantly expand the translational potential of recombinant collagen, particularly for tissue engineering, regenerative medicine, and implantable biomaterials.

4.1.3. Evaluation of triple-helical structure

The stability of the triple helix is a critical determinant of the biological functionality, structural integrity, and translational potential of recombinant collagen. Among available analytical approaches, circular dichroism (CD) spectroscopy remains the most widely used technique. Its characteristic far-UV signature (190–250 nm)—featuring a positive peak near 220 nm and a negative peak around 198 nm—serves as a definitive indicator of triple-helical structure. Temperature-dependent CD scans enable determination of the melting temperature (T_m), facilitating quantitative comparisons of thermal stability across collagens from different sources or with distinct PTMs [12,173,174].

Differential scanning calorimetry (DSC) provides complementary thermodynamic insights, directly measuring T_m and enthalpic changes during denaturation to reveal folding energetics and inform structural optimization [172,175]. Fourier transform infrared (FTIR) spectroscopy further supports structural analysis by detecting shifts in the amide I (∼1650 cm⁻¹) and amide II (∼1550 cm⁻¹) bands, which reflect hydrogen-bond integrity and secondary structure organization [26]. At the supramolecular level, small-angle X-ray scattering (SAXS) offers information on global conformational parameters such as helix diameter and axial repeat distance [176], while atomic force microscopy (AFM) enables nanoscale visualization of molecular morphology and fibrillar assembly patterns [177]. Together, these complementary techniques enable systematic evaluation of triple-helix formation, stability, and molecular design efficacy, providing a robust analytical framework that underpins both fundamental research and the translational development of recombinant collagen-based biomaterials.

4.2. Non-triple helical recombinant collagen

In expression systems that lack the molecular machinery for triple-helix assembly, collagen is frequently produced as single-chain gelatin-like proteins (GLPs). These GLPs, composed of modular repeats of the Gly-Xaa-Yaa tripeptide, have been expressed in E. coli, yeast, and mammalian cells. Werten et al. utilized the S. cerevisiae α-mating factor signal peptide to achieve efficient secretion of non-hydroxylated gelatin derived from murine type I and rat type III collagen sequences [178], and subsequently enhanced the hydrophilicity of GLPs through amino acid composition optimization [179]. Olsen et al. reported high-level expression of human-derived gelatin with remarkable molecular homogeneity [180], while other studies have demonstrated the expression of soluble collagen fragments in yeast [181,182]. Additionally, GLPs composed of repetitive Gly-Pro-Pro motifs have been produced [87], and an artificial gelatin constructed from tandem 30–amino-acid units derived from type I collagen has also been reported [101].

Unlike conventional denatured gelatin—which is heterogeneous, animal-derived, and compositionally variable—recombinant GLPs are structurally defined, highly reproducible, and tunable in both mechanical and biological properties [183]. Their modular architecture also enables the precise incorporation of bioactive motifs such as RGD and GFOGER without the structural constraints imposed by a full triple helix. However, the absence of a triple-helical backbone limits their ability to form fibrils and significantly reduces mechanical reinforcement, restricting their utility in load-bearing tissue engineering applications.

4.3. Fusion of recombinant collagen

Fusion strategies integrate bioactive motifs or heterologous protein domains into the collagen backbone, enabling precise modulation of physicochemical properties, bioactivity, and environmental responsiveness. This modular design greatly broadens the functional landscape of recombinant collagens, unlocking advanced applications in tissue engineering, regenerative medicine, and bioactive scaffolding. Hayashi et al. successfully fused epidermal growth factor (EGF) and interleukin-2 (IL-2) to the triple-helical domain of human type III collagen, preserving both the structural integrity of the helix and the bioactivity of the fused proteins [184,185]. Chen et al. incorporated human bone morphogenetic protein-2 (hBMP-2) into a human-like collagen (HLC) backbone, generating an osteoinductive construct that promoted osteogenesis [186]. Through site-directed mutagenesis, integrin- and heparin-binding motifs have been precisely incorporated, significantly enhancing cell adhesion [18].

Fusion with functional fragments of hyaluronic acid and chondroitin sulfate has been shown to promote chondrogenic differentiation of human mesenchymal stem cells (hMSCs), thereby improving the bioactivity and suitability of collagen scaffolds for cartilage tissue engineering [17] (Fig. 4A). Hu et al. linked short peptides (KISALKE)₃ and (EISALEK)₃ to collagen-like domains, yielding triple-helical structures that formed hydrogels with excellent cytocompatibility and robust support for NIH 3T3 fibroblast proliferation [187] (Fig. 4B). Incorporating silk fibroin sequences into collagen enhanced mechanical strength and material affinity [19], while chimeric collagens bearing integrin- and fibronectin-binding motifs improved hMSC adhesion and proliferation, offering promising routes for tissue-specific scaffold design (Fig. 4C). Collagen–antimicrobial peptide fusions combined structural reinforcement with antimicrobial activity, making them attractive candidates for wound healing applications. Moreover, engineered crosslinking motifs have enabled precise control over hydrogel stiffness, while photosensitive domains allow spatiotemporal regulation of scaffold mechanics, further expanding the functional versatility of fusion-based collagen biomaterials.

Fig. 4.

Structural and functional design strategies for recombinant fusion collagens. (A) Incorporation of hyaluronic acid– and chondroitin sulfate–binding peptide motifs into the collagenous domain to enhance chondrogenic bioactivity. Reproduced with permission [17]. Copyright 2016, Wiley-VCH. (B) Fusion of hydrophobic peptide sequences at the termini of collagen to promote self-assembly into anisotropic, self-supporting hydrogels. Reproduced with permission [174]. Copyright 2021, Amer Chemical Soc. (C) Integration of silk fibroin fragments and integrin-binding sites within the collagen backbone to improve mechanical strength and enhance cell adhesion. Reproduced with permission [19]. Copyright 2013, Elsevier.

Despite their promise, fusion designs present potential risks, including immunogenicity, misfolding, and altered degradation kinetics [26,188,189]. Advances in AI-driven sequence optimization and molecular dynamics simulations may enable accurate prediction of epitope distribution, rational balancing of functionality with structural stability, and early-stage safety profiling [190,191]. Future development of fusion-type recombinant collagens will require careful integration of functional innovation with rigorous biosafety assessment, leveraging interdisciplinary design strategies to accelerate their translation into clinically viable biomaterials.

Taken together, the three major classes of recombinant collagen represent a continuum from structural biomimicry to functional innovation. Triple-helical constructs most faithfully recapitulate the architecture and mechanical properties of native fibrils, while gelatin-like proteins (GLPs) and other non-helical variants prioritize solubility, tunability, and ease of processing. Fusion collagens, in contrast, exemplify rational molecular engineering, enabling the integration of bioactive motifs and multifunctional domains. The selection among these formats is inherently application-driven, spanning structural scaffolds, injectable therapeutics, and next-generation smart biomaterials designed for precision regenerative medicine (Table 3).

Table 3.

Categories of recombinant collagen and their key characteristics.

Category	Structural features	Advantages	Limitations	Applications
Triple-helical collagen	Right-handed triple helices composed of repetitive Gly-Xaa-Yaa motifs	Native-like fibrillogenesis; high bioactivity; enhanced mechanical strength	Low yield; insufficient hydroxylation; complex folding process	Structural scaffolds; ECM-mimetic matrices; load-bearing hydrogels
Non-triple-helical collagen-like proteins (GLPs)	Defined Gly-Xaa-Yaa repeats without triple-helical conformation	High solubility; modular and tunable design	Weak mechanical strength; limited fibrillogenesis; compromised structural integrity	Soft tissue scaffolds; cosmetic and nutraceutical formulations
Fusion collagens	Collagen domains covalently linked to functional motifs	Multifunctionality; tunable mechanical and bioactive properties; enhanced cell–matrix interactions	Complex design and manufacturing; potential immunogenicity; regulatory hurdles	Smart biomaterials; bioactive scaffolds; targeted regenerative therapeutics

4.4. AI-assisted design and modeling

Recent advances in deep learning (DL) and molecular dynamics (MD) simulations are revolutionizing the rational design of recombinant collagen. Unlike conventional sequence optimization approaches that rely largely on empirical heuristics, AI-driven methods can extract the underlying principles of triple-helix stability, molecular folding, and supramolecular assembly from large-scale sequence–function datasets [192]. When coupled with genetic algorithms (GA), active learning, and evolutionary optimization, these approaches enable highly efficient and iterative sequence refinement [193].

Specialized AI models have demonstrated powerful capabilities in predicting structure–function relationships [194,195]. For instance, CollagenTransformer, built on a natural language processing (NLP) framework, can predict the T_mof triple helices in an end-to-end manner, providing a high-throughput and cost-effective platform for candidate sequence screening [196]. AlphaFold Multimer achieves prediction accuracies of 67 % and 69 % for heteromeric and homomeric complexes, respectively, significantly improving structural modeling precision [197,198]. In parallel, geometric deep learning and generative models such as ProteinMPNN, ESM-IF, and ColDiff are enabling de novo sequence design, showing strong concordance with experimental results in predicting triple-helix formation and thermal stability [199,200]. Integrative frameworks that combine generative AI with genetic algorithms—such as ColGen-GA—can now generate thousands of candidate sequences within hours, achieving T_m prediction errors below 5 % and far exceeding the throughput of conventional MD simulations [193]. Moreover, reinforcement learning and modular design strategies allow the incorporation of functional peptide motifs while preserving triple-helical and fibrillar architectures, thereby enabling engineered collagens with tailored biological activities [75,[201], [202], [203]].

Future research should prioritize the development of standardized sequence–function datasets, uncertainty quantification frameworks, and multi-objective optimization algorithms. By integrating AI, MD simulations, and automated experimental platforms into a closed-loop design–build–test–learn (DBTL) cycle, the transition from molecular design to industrial translation can be substantially accelerated [204]. Within this emerging paradigm, AI will evolve beyond a predictive tool to become the central engine of collagen engineering—orchestrating simultaneous optimization of thermal stability, immunogenicity, self-assembly dynamics, and secretion efficiency. Such integration promises to unlock a new era of precision-designed collagen biomaterials tailored for regenerative medicine and clinical applications.

5. Application of recombinant collagen biomaterials

5.1. Bone regeneration

Recombinant collagen-based scaffolds offer exceptional design flexibility, enabling precise modulation of crosslinking density, mineralization level, and bioactive component integration. Such tunability allows for controlled degradability aligned with the bone healing timeline while maintaining an optimal balance between mechanical strength and biological activity. Incorporation of synthetic polymers—renowned for their mechanical robustness and elasticity—further enhances scaffold resilience and structural integrity [205]. Their synergistic combination with recombinant collagen couples mechanical reinforcement with intrinsic bioactivity, thereby establishing a pro-regenerative microenvironment that supports osteogenesis.

Wang et al. engineered a polyethylene glycol (PEG)–chondroitin sulfate (ChS)–triple-helical recombinant collagen (THRC) hydrogel featuring an interconnected porous architecture, high mechanical strength, low swelling ratio, and controlled degradation profile. This composite scaffold significantly enhanced the proliferation, migration, and osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs) [206] (Fig. 5A). Similarly, human recombinant type I collagen (rhCol-I) was grafted onto poly(L-lactic acid) (PLLA) membranes via alkaline hydrolysis and Schiff base chemistry, producing a bioactive PLLA–rhCol composite that promoted angiogenesis and markedly accelerated bone repair in vivo [207] (Fig. 5B).

Fig. 5.

Recombinant collagen–based composite scaffolds for bone defect repair. (A) A highly bioactive hydrogel (PEG–ChS–THRC) composed of polyethylene glycol (PEG), chondroitin sulfate (ChS), and triple-helical recombinant collagen (THRC) promotes robust bone tissue regeneration. Reproduced with permission [206]. Copyright 2024, Springernature; (B) A composite scaffold of poly(L-lactic acid) (PLLA) grafted with recombinant human type I collagen (rhCol-I) enhances osteogenic performance and accelerates in vivo bone healing. Reproduced with permission [207]. Copyright 2024, Elsevier. (C) A freeze-dried scaffold (TRFS) incorporating transforming growth factor-β3 (TGF-β3), recombinant human collagen (RHC), and chondroitin sulfate (CS), combined with human periodontal ligament stem cells (hPDLSCs), significantly promotes osteogenic differentiation and bone regeneration at defect sites. Reproduced with permission [213]. Copyright 2021, Frontiers Media SA.

Growth factors play a pivotal role in bone regeneration by orchestrating cell proliferation, lineage commitment, and extracellular matrix synthesis. Recombinant collagen serves as an efficient and biocompatible carrier for their controlled delivery, enabling spatiotemporal regulation of osteogenic signaling and thereby enhancing bone formation and accelerating defect repair [208,209]. A fusion construct comprising human-like collagen and bone morphogenetic protein-2 (HLC–hBMP2) significantly enhanced mesenchymal stem cell (MSC) adhesion and migration, exhibited controlled in vivo degradation to sustain osteoinductive activity, and precisely modulated BMP-2 release to improve bioavailability. In a rat calvarial defect model, this fusion system markedly promoted new bone formation without eliciting inflammatory responses [186].

Building upon this strategy, He et al. developed a BMP-2–loaded hydrogel by crosslinking triple-helical recombinant collagen (THRC) with oxidized carboxymethyl cellulose (OCMC) and N-succinyl chitosan (NSC), which significantly enhanced bone regeneration in rat calvarial defects [210]. Recombinant collagen peptide (RCP) microspheres derived from type I collagen have also been utilized as BMP-2 carriers, and when combined with polysaccharides, effectively induced ectopic bone formation [211]. Guo et al. reported a recombinant human collagen (rhCol)/basic fibroblast growth factor (bFGF) hydrogel crosslinked with transglutaminase (TG), which enabled sustained release of bFGF, upregulated osteogenesis-related protein expression, and accelerated bone repair [212]. Furthermore, a freeze-dried TGF-β3/RHC/CS (TRFS) scaffold seeded with human periodontal ligament stem cells (hPDLSCs) significantly enhanced osteogenic gene expression and differentiation, resulting in effective cranial defect healing [213] (Fig. 5C).

To overcome the limitations of recombinant collagen in mechanical strength and mineralization capacity, composite strategies incorporating inorganic bone substitutes have garnered significant attention. The integration of osteoconductive materials such as hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) enhances the mechanical integrity and structural stability of collagen-based scaffolds while simultaneously augmenting their osteogenic potential. Such hybrid systems provide a promising platform for biomimetic repair of large bone defects, recapitulating the hierarchical composition of native bone.

For instance, RCP/β-TCP composite scaffolds fabricated via thermal crosslinking and freeze-drying demonstrated significantly enhanced new bone formation in vivo [214]. A biomimetic nHA/RHLC/PLA scaffold, designed with an organic-to-inorganic ratio closely mimicking natural bone, achieved complete replacement by newly formed bone within 24 weeks post-implantation, resulting in effective defect repair [215]. Incorporation of recombinant human BMP-2 (rhBMP-2) into such composite scaffolds further increased osteoinductive potential and accelerated bone regeneration [216]. Additionally, polydopamine (pDA)–based surface modification was employed to deliver a BMP-2–derived peptide (P24), which further enhanced osteogenesis and upregulated bone-specific gene expression [217].

Direct blending of collagen with inorganic components frequently leads to phase separation, resulting in heterogeneous distribution and poor interfacial integration. In contrast, natural bone features a hierarchically organized matrix in which collagen fibrils are intimately co-aligned with nanoscale hydroxyapatite (nHA), forming a unique mineralized collagen architecture that underpins its mechanical strength and biological functionality. Recapitulating this architecture is therefore essential to faithfully reproduce the microscopic composition and structural organization of native bone and to achieve superior biomimetic performance.

To this end, He et al. employed recombinant collagen as a biomimetic template to direct the in situ nucleation and growth of nanostructured mineralized collagen, followed by fabrication of a three-dimensional porous scaffold composed of mineralized recombinant collagen and sodium alginate (MRCSA). The resulting scaffold closely mimics native bone in both organic–inorganic composition and nanoscale architecture, exhibiting excellent biocompatibility, controlled degradability, and enhanced cellular activity and osteoinductive potential [218] (Fig. 6A). Similarly, a biomimetic collagen–hydroxyapatite composite scaffold (CCMH) was fabricated via unidirectional freeze-casting combined with in situ peristaltic mineralization. This scaffold accurately recapitulates the composition and anisotropic channel architecture of native bone, demonstrating excellent cytocompatibility and bioactivity and markedly enhancing new bone tissue regeneration [219] (Fig. 6B).

Fig. 6.

Recombinant collagen–nano-hydroxyapatite composite scaffolds for enhanced bone regeneration. (A) A three-dimensional porous scaffold composed of mineralized recombinant collagen and sodium alginate (MRCSA), which biomimics the organic–inorganic composition and nanoscale architecture of native bone, effectively promoting new bone formation and complete defect repair. Reproduced with permission [218]. Copyright 2024, Elsevier. (B) A biomimetic scaffold composed of a collagen composite matrix and hydroxyapatite (CCMH), closely replicating the composition and anisotropic channel structure of native bone, thereby significantly accelerating bone tissue regeneration. Reproduced with permission [219]. Copyright 2023, Elsevier.

5.2. Cartilage repair

Recombinant collagen has shown significant promise in cartilage tissue engineering, particularly when combined with seed cells, polysaccharides, and bioactive factors to construct biomimetic scaffolds that closely replicate the native extracellular matrix (ECM). Such systems have demonstrated robust regenerative efficacy and strong translational potential. Recombinant collagen hydrogels seeded with autologous chondrocytes can effectively support cartilage regeneration. Pulkkinen et al. implanted recombinant human type II collagen (rhCII) gels containing rabbit chondrocytes into osteochondral defects, resulting in complete defect filling with cartilage rich in type II collagen and proteoglycans after six months [220]. Similarly, rhCII/PLA scaffolds seeded with chondrocytes promoted the formation of hyaline-like cartilage in miniature pigs within four months [221].

Natural polysaccharides, known for their excellent biocompatibility and biodegradability [222], can form ECM-mimetic hydrogels when combined with recombinant collagen, further enhancing their regenerative performance. Xu et al. developed a photocrosslinked hydrogel (HA–rhCol III) by integrating methacrylated hyaluronic acid (HA-MA) with recombinant human type III collagen (rhCol III), yielding a porous lamellar structure [223] (Fig. 7A). In co-culture with chondrocytes, the hydrogel maintained structural integrity while significantly promoting cell adhesion, proliferation, and synthesis of DNA and glycosaminoglycans (GAGs). In a rabbit cartilage defect model, implantation led to seamless tissue integration and complete defect filling with newly formed cartilage after 12 weeks. Xie et al. constructed a composite hydrogel scaffold (HLC–PVA–HA) via a freeze–thaw approach, which exhibited high mechanical strength, a well-organized porous microstructure, and excellent chondrogenic activity in vivo [224].

Fig. 7.

Applications of recombinant collagen-based biomaterials in cartilage defect repair. (A) A photo-crosslinkable hydrogel (HA–rhCol III) composed of methacrylated hyaluronic acid (HA-MA) and recombinant human type III collagen (rhCol III) maintained structural integrity, enhanced chondrocyte activity, and significantly promoted cartilage regeneration. Reproduced with permission [223]. Copyright 2025, Elsevier. (B) Composite microgel granules (SHM 3 %Ce/3 %Se), consisting of cerium/selenium co-doped mesoporous bioactive glass, sodium alginate, and recombinant collagen, exhibited excellent repair efficacy in a rabbit cartilage defect model. Reproduced with permission [225]. Copyright 2023, Tsinghua Univ Press. (C) A 3D-printed photo-crosslinkable composite hydrogel (CF/CM/3 %LAP/KGN) incorporating recombinant collagen, chitosan, Laponite-XLG nanoclay, and Kartogenin (KGN)-loaded nanoparticles enabled sustained KGN release, robustly induced chondrogenic differentiation, and effectively promoted cartilage tissue regeneration. Reproduced with permission [226]. Copyright 2024, Elsevier.

Functionalization with bioactive molecules further enhances the therapeutic efficacy of recombinant collagen scaffolds. Fan et al. designed a composite microgel graft (SHM 3 %Ce/3 %Se) comprising cerium- and selenium-co-doped mesoporous bioactive glass, sodium alginate, and recombinant collagen. In a rabbit cartilage defect model, this graft facilitated seamless integration between regenerated tissue and native cartilage, producing a smooth and well-contoured surface while improving bone regeneration parameters after 12 weeks [225] (Fig. 7B). Similarly, a 3D-printable composite hydrogel (CF/CM/3 %LAP/KGN), composed of recombinant collagen, chitosan, nanoscale Laponite-XLG clay, and kartogenin (KGN)-loaded nanoparticles, enabled sustained KGN release and significantly enhanced hBMSC chondrogenesis, thereby supporting cartilage regeneration and functional restoration of defect sites [226] (Fig. 7C).

Moreover, biomimetic bilayer scaffolds have emerged as a powerful approach for osteochondral tissue engineering, enabling the structural and functional reconstruction of the cartilage–bone interface. In such designs, the bone layer is composed of a composite of human-like collagen (HLC), hyaluronic acid (HA), and hydroxyapatite (HAp), while the cartilage layer consists of a porous HLC/HA matrix [227]. These scaffolds exhibit a continuous lamellar architecture, optimal pore size distribution, high porosity, and favorable mechanical properties, thereby supporting hBMSC adhesion, proliferation, and lineage-specific differentiation. In osteochondral defect models, they enhanced collagen and GAG deposition and promoted the formation of hyaline-like tissue, underscoring their strong potential for functional cartilage regeneration.

5.3. Skin repair and regeneration

Recombinant collagen–based hydrogels have emerged as a transformative platform for skin repair and regeneration. By integrating antimicrobial agents, growth factors, metal ions, natural polymers, stem cells, and exosomes, these systems achieve smart responsiveness, controlled release, and self-repair capabilities—enabling precise modulation of the wound microenvironment, attenuation of inflammation, stimulation of angiogenesis, and acceleration of tissue regeneration. Ye et al. demonstrated that recombinant type III collagen (rCol III) enhanced fibroblast activity, reduced transepidermal water loss (TEWL), and promoted collagen regeneration in UV-damaged skin [228]. Yang et al. reported that a triple-helical recombinant collagen (THRC) hydrogel improved antioxidant capacity and dermal architecture during sunburn repair [229]. Liu et al. further engineered recombinant human type I collagen and CF-1552(I), which not only accelerated wound closure but also exhibited potent antioxidant activity and facilitated ECM remodeling in photoaging models [230]. Electrospun rhCol III nanofiber membranes (rhCol III EN NF) additionally provide robust mechanical strength and strong regenerative potential [231].

In aesthetic dermatology, recombinant collagen hydrogels have gained traction as next-generation injectable fillers. Tetra-hydroxymethyl phosphonium chloride (THPC)-crosslinked recombinant collagen hydrogels (TH-THRC) exhibited excellent injectability, mechanical stability, and bioactivity, enhancing fibroblast adhesion, proliferation, and migration. In photoaging models, TH-THRC increased dermal density, elasticity, and collagen deposition while reducing TEWL [232]. Moreover, sustained administration of rhCol III for eight weeks in UV-induced skin injury models mitigated structural damage, promoted type I and III collagen secretion, and remodeled the ECM [233].

Co-assembly of recombinant collagen with natural polymers further enhances structural complexity and functional performance. Hydrogels formed by co-assembling rhCol III and chitosan (CS) rapidly gel, provide excellent wound coverage, exhibit strong antibacterial properties, and promote fibroblast adhesion and migration, thereby accelerating full-thickness skin wound healing (Fig. 8A) [234]. Thermoresponsive RHC/CS hydrogels quickly form barrier structures at body temperature, adapting to the highly permeable burn wound environment and further promoting angiogenesis and tissue regeneration [235]. Hydrogels generated through covalent crosslinking of thiolated hyaluronic acid (HA) with rhCol III regulate the type I/III collagen ratio, enhance neovascularization and collagen deposition, and improve wound reconstruction quality (Fig. 8B) [236]. Additionally, gelatin–rhCol III composite hydrogels exhibit excellent cell compatibility and tissue integration, further broadening the scope of recombinant collagen in skin regeneration [237].

Fig. 8.

Recombinant collagen–natural polymer composite hydrogels for skin repair. (A) A composite hydrogel composed of recombinant type III collagen (rhCol III) and chitosan (CS) exhibits excellent antibacterial activity and effective wound coverage, thereby accelerating skin healing. Reproduced with permission [234]. Copyright 2023, Royal Soc Chemistry; (B) A composite hydrogel formed by recombinant type III collagen (rhCol III) and thiolated hyaluronic acid (HS) promotes angiogenesis, modulates inflammation, and enhances tissue regeneration. Reproduced with permission [236]. Copyright 2024, Royal Soc Chemistry.

Incorporation of functional proteins into recombinant collagen hydrogels significantly enhances their immunomodulatory and regenerative performance. For example, RhFN–RHC hydrogels facilitate fibroblast adhesion and migration, accelerate epithelial regeneration, and modulate the immune microenvironment to suppress inflammation and promote epidermal repair [238]. Similarly, G4–RHC hydrogels incorporating guanosine quadruplexes enable sustained release of recombinant collagen (RHC), induce M2 macrophage polarization, stimulate angiogenesis, and markedly accelerate wound healing [239].

Advanced crosslinking strategies and fabrication techniques have further reinforced the structural integrity and mechanical resilience of recombinant collagen hydrogels, enhancing their performance in complex wound environments. GelMA–RC hydrogels synthesized via EDC/NHS crosslinking exhibit improved mechanical strength, upregulated keratin and collagen expression, and accelerated epithelial regeneration [240]. Enzyme–chemical dual-crosslinked HLC–carboxymethyl chitosan (CCS) scaffolds combine excellent injectability with a porous structure that facilitates cellular infiltration and dermal remodeling [241]. Additionally, an HLC/HA/CCS composite hydrogel crosslinked by transglutaminase forms a highly interconnected porous network with strong antibacterial properties and high fibroblast affinity, effectively promoting wound closure and reducing scar formation in deep second-degree burn models [242].

Stem cell–based strategies further expand the therapeutic potential of recombinant collagen in treating chronic and refractory wounds. A photocrosslinkable hADSCs@rHCIII-MA hydrogel—composed of methacrylated recombinant human type III collagen (rHCIII-MA) and human adipose-derived stem cells (hADSCs)—creates a stable 3D microenvironment that supports stem cell adhesion, proliferation, and metabolism, while prolonging cell retention and enhancing immunomodulatory and pro-angiogenic activity in diabetic wound models [243] (Fig. 9A). Recombinant collagen hydrogels incorporating stem cell–derived extracellular vesicles (EVs), such as the rhCol III–EVs composite, enable sustained exosome release, drive macrophage polarization toward the M2 phenotype, suppress inflammation, and enhance angiogenesis and tissue repair [244]. A genipin-crosslinked rhCol I/CMC–Exos hydrogel, prepared from recombinant type I collagen, carboxymethyl chitosan, and umbilical cord mesenchymal stem cell exosomes, exhibits excellent biocompatibility, controlled exosome release, and robust promotion of dermal regeneration and neovascularization [245] (Fig. 9B). Moreover, a rhCol III/sodium alginate (SA)/EV composite hydrogel designed for localized exosome delivery demonstrates potent antioxidative, anti-inflammatory, and regenerative effects, highlighting its promise as a precision therapy platform for complex wound healing scenarios [246].

Fig. 9.

Multifunctional recombinant collagen-based hydrogels for chronic wound healing. (A) A photocrosslinkable hydrogel composed of human adipose-derived stem cells encapsulated within recombinant human type III collagen (hADSCs@rHCIII) prolongs stem cell retention, enhances immunomodulatory and pro-angiogenic activities, and accelerates wound closure, thereby promoting diabetic wound repair. Reproduced with permission [243]. Copyright 2024, Wiley-VCH. (B) A recombinant human type I collagen/sodium carboxymethyl cellulose–exosome (rhCol I/CMC–Exos) hydrogel supports sustained exosome release, facilitating dermal regeneration and neovascularization. Reproduced with permission [245]. Copyright 2024, Elsevier. (C) A dopamine-modified hyaluronic acid/recombinant collagen (HA-DA@rhCol) hydrogel synergistically enhances cell migration, angiogenesis, inflammation regulation, and collagen deposition, thereby expediting wound healing. Reproduced with permission [249]. Copyright 2024, KEAI; (D) A multifunctional CS/rCol I/Cu/DMA hydrogel, integrating a metal–polyphenol network with chitosan, exhibits potent antibacterial activity, self-healing capability, hemostatic function, and pro-angiogenic effects. Reproduced with permission [251]. Copyright 2023, MDPI.

Multifunctional hydrogels provide advanced therapeutic strategies for chronic and infected wounds. A microenvironment-responsive recombinant type III collagen (rhCol III) hydrogel embedded with PDA@Ag nanoparticles exhibited potent antibacterial, antioxidative, and anti-inflammatory effects while enhancing the expression of bFGF and VEGF, promoting collagen deposition, and stimulating neovascularization [247]. A rhCol III-based hydrogel incorporating antimicrobial peptides and perfluoronaphthalene vesicles effectively eradicated bacterial infections at wound sites [248]. Dopamine-modified hyaluronic acid combined with rhCol III yielded the HA-DA@rhCol hydrogel, characterized by a well-defined porous structure, high swelling capacity, tunable degradability, strong tissue adhesion, antioxidative activity, and photothermal responsiveness [249] (Fig. 9C). In diabetic wound models, this hydrogel significantly enhanced cell migration, angiogenesis, inflammation resolution, and collagen remodeling, thereby accelerating wound closure.

Similarly, an SA@MnO₂/RHC/MSC hydrogel integrating antioxidative nanoparticles with stem cells promoted epithelial regeneration, collagen deposition, and neovascularization in diabetic wounds [250]. A CS/rCol I/Cu/DMA hydrogel formed via metal–polyphenol coordination exhibited multifunctional properties—including antibacterial activity, self-healing capacity, hemostasis, and pro-angiogenic effects—markedly accelerating granulation tissue formation and wound closure in full-thickness defect models [251] (Fig. 9D). Moreover, a rhCol III-based microneedle hydrogel incorporating naproxen-loaded PLGA nanoparticles enabled minimally invasive delivery and sustained release, effectively modulating inflammation, enhancing cell migration and angiogenesis, and improving wound healing outcomes in diabetic models [252].

Tissue-engineered skin equivalents (TESE) represent a promising regenerative strategy, enabling structural reconstruction and functional restoration of damaged skin. A TESE constructed by seeding fibroblasts into a recombinant humanized collagen/transglutaminase (rHC/TG) hydrogel enhanced full-thickness wound repair and exhibited excellent cellular activity [253]. A stratified skin graft (STSG) fabricated from rhCol III powder demonstrated favorable water retention and degradation kinetics, significantly accelerating wound closure, neovascularization, basement membrane formation, and dermal regeneration while reducing scar formation [254]. Furthermore, a composite dressing composed of recombinant collagen, micronized acellular dermal matrix (mADM), and defined type I/III collagen ratios provided excellent flexibility, conformability, and porosity, maintained a moist wound microenvironment, prevented exudate accumulation, and promoted fibroblast adhesion, angiogenesis, and appendage regeneration in vivo [255].

Clinical translation of recombinant collagen is accelerating, with several products already approved. The Vergenix™ series (Vergenix™ WD, Vergenix™ FG, and Vergenix™ STR), derived from plant-based rhCollagen, has received CE certification for chronic wound care and soft tissue regeneration [256,257]. Recombinant humanized type III collagen lyophilized fiber has been approved by the National Medical Products Administration (NMPA) in China for clinical use in wound repair and tissue regeneration. In parallel, a range of Class II medical devices based on humanized recombinant collagen has been developed and widely applied in wound dressings and aesthetic tissue restoration.

By contrast, animal-derived collagens remain more clinically established, with products available as microfibers, membranes, granules, hydrogels, and sponges for treating surgical incisions, radiation injuries, diabetic wounds, and chronic ulcers. Representative examples include Catrix® bovine collagen powder and Collatek® bovine collagen hydrogel, widely used for refractory ulcers and full-thickness skin defects. In China, several Class III animal-derived collagen dressings have also received regulatory approval. Overall, while animal-derived collagen continues to dominate due to its mature manufacturing processes and broad clinical adoption, recombinant collagen—characterized by high batch-to-batch consistency, low immunogenicity, and tunable functionality—is rapidly emerging as a next-generation material platform for advanced skin repair and regenerative medicine (Table 4).

Table 4.

Comparison of recombinant and animal-derived collagen in skin repair materials.

Category	Recombinant Collagen	Animal-Derived Collagen
Source	Genetic engineering expression systems (E. coli, yeast, plants, mammalian cells, etc.)	Animal tissues (bovine, porcine, piscine, etc.)
Representative Products	Vergenix™ WD, Vergenix™ FG, Vergenix™ STR (CE-certified); recombinant humanized type III collagen lyophilized fiber (NMPA-approved)	Catrix® (bovine collagen powder), Collatek® (bovine collagen hydrogel); Collagen dressings (Class III medical devices)
Regulatory Approvals	CE-certified in Europe; multiple Class II medical devices approved by the NMPA in China	FDA-approved in the U.S.; CE-certified in Europe; multiple NMPA-approved products in China
Applications	Wound repair; aesthetic filling.	Acute wound management (surgical incisions, lacerations, burns, radiation injuries); chronic ulcer treatment (diabetic, venous, pressure ulcers); tissue regeneration
Advantages	High batch-to-batch consistency; low immunogenicity; potential for programmable functionalities	Mature manufacturing technologies; abundant raw materials; relatively low production cost
Limitations	Limited yield; high production cost; clinical translation still ongoing	Batch-to-batch variability; potential immunogenicity and pathogen transmission risks

5.4. Cornea regeneration

Recombinant collagen, owing to its excellent biocompatibility, optical properties, and versatile processability, has emerged as a promising matrix for corneal tissue engineering. Efforts to enhance transparency, replicate native stromal microarchitecture, and guide cell behavior are accelerating its progress toward clinical translation. Li et al. fabricated a transparent corneal substitute by crosslinking recombinant human type I and type III collagens withEDC and NHS [258]. Building on this, hydrogels composed of recombinant human type I collagen (RHCI) with distinct fiber organizations—random (rRHCI) or aligned (aRHCI)—were prepared [259]. The rRHCI hydrogel exhibited superior optical transparency and mechanical strength, effectively attenuating inflammation and promoting epithelial, stromal, and nerve regeneration, whereas the aRHCI variant, with thicker fibers and reduced transparency, did not meet the stringent optical requirements for corneal transplantation.

To better recapitulate native corneal architecture, Sun et al. chemically modified recombinant collagen with methacrylic anhydride to generate a photocrosslinkable hydrogel (MI-RHCMA) featuring microgrooved and inverse opal–like nanostructures [260] (Fig. 10A). This biomimetic topography guided limbal stromal stem cell alignment and keratocyte differentiation, significantly improving regenerative outcomes. In vivo, MI-RHCMA supported robust stromal repair. Zhao et al. further developed a stromal replacement corneal patch (SRCP) by integrating a decellularized corneal nanotubular scaffold with recombinant human collagen and methacrylated gelatin [261] (Fig. 10B). At physiological temperature (37 °C), SRCP enabled sustained gelatin release and could be photoactivated with 405 nm light to achieve stable stromal integration. In vivo implantation accelerated epithelial regeneration and visual recovery within two weeks and maintained stromal stability without scarring for up to four weeks. Together, these studies highlight the potential of recombinant collagen to serve as a biomimetic, cell-instructive matrix for corneal regeneration, offering tunable mechanical properties, optical clarity, and biological activity essential for next-generation corneal implants and vision-restorative therapies.

Fig. 10.

Recombinant collagen-based biomaterials for corneal repair. (A) MI-RHCMA hydrogel patch featuring aligned microgrooves and inverse opal-like nanostructures for effective corneal injury repair. Reproduced with permission [260]. Copyright 2022, Elsevier. (B) Biomimetic stromal replacement corneal patch (SRCP) exhibiting ultrastructural resemblance to native cornea and superior optical transparency. Reproduced with permission [261]. Copyright 2025, Elsevier.

5.5. Other biomedical applications

Recombinant collagen exhibits broad therapeutic potential beyond classical musculoskeletal and dermal contexts, demonstrating efficacy in gynecology, cardiovascular medicine, oncology, and oral health. In gynecological applications, an injectable, self-assembling alginate–recombinant collagen hydrogel enhanced endometrial stromal cell proliferation and restored endocrine homeostasis, thereby accelerating endometrial regeneration [262], while a rhCol III–HA) composite hydrogel prolonged intrauterine retention and improved therapeutic efficacy [263]. In cardiovascular medicine, recombinant collagen has been explored as a next-generation biomaterial to overcome the limitations of glutaraldehyde-crosslinked bioprosthetic heart valves (BHVs), which are prone to thrombosis, calcification, and chronic inflammation [264]. Integration of rhCol III with a metal–phenolic network enhanced antithrombotic performance, endothelialization, and anti-calcification properties [265], while a fucoidan–rhCol III coating further improved hemocompatibility and mitigated calcification [266].

Zhou et al. fabricated artificial valves using decellularized porcine pericardium, rhCol III, and glycidyl methacrylate, achieving robust mechanical strength, anticoagulant activity, and attenuated inflammatory responses [267]. Additionally, rhCol III coatings on PLA scaffolds promoted endothelialization, suppressed inflammation, and limited neointimal hyperplasia, thereby conferring strong antithrombotic efficacy [268]. In oncology, rhCol III was found to inhibit glutathione S-transferase P1 (GSTP1) expression and autophagy, demonstrating promising antitumor activity [269]. In oral medicine, recombinant humanized type XVII collagen (rhCol XVII) significantly stimulated gingival fibroblast proliferation and migration, accelerating oral ulcer healing [270]. Collectively, these findings underscore recombinant collagen's multifaceted bioactivity and translational promise across diverse disease contexts, positioning it as a versatile platform for precision regenerative therapies.

Beyond biomedicine, recombinant collagen is gaining traction in the food industry as both a structural scaffold for cellular agriculture and a functional additive to improve texture, palatability, and nutritional value [271]. Collagen peptides are already incorporated into beverages, dairy, and meat products to enhance structural properties and nutritional content [272,273]. Moreover, oral supplementation with collagen peptides has been shown to stimulate elastin synthesis and suppress MMP-1/3 secretion, thereby reducing elastin degradation and maintaining dermal extracellular matrix homeostasis, highlighting their potential as dietary supplements for skin health [274].

Taken together, these studies emphasize the versatility of recombinant collagen platforms—spanning triple-helical constructs, human-like variants, and type-specific isoforms—and their integration with polymers, polysaccharides, inorganic minerals, and bioactive molecules(Table 5). Such modularity enables fine-tuning of mechanical, biochemical, and degradation properties to support applications across bone, cartilage, skin, cornea, and soft tissue regeneration. Moreover, their adaptability into diverse formats—including hydrogels, scaffolds, sponges, microspheres, and 3D-printed architectures—positions recombinant collagen as a next-generation multifunctional biomaterial bridging fundamental research with clinical translation.

Table 5.

Representative recombinant collagen-based constructs and their potential applications in tissue repair and regenerative medicine.

Collagen Construct	Expression System	Experimental Model	Biomaterial Preparation	Application Prospects	Ref.
Triple-helical recombinant collagen (THRC)	E. coli	Rat; Mouse	Composites with polymers, polysaccharides, minerals, and crosslinkers; collagen solutions and gels	Bone and cartilage regeneration, skin repair	[206,210,218,219,229,232]
Human-like collagen (HLC)	P. pastoris; CHO	Rat; Rabbit	Fusion proteins, composite hydrogels, microgranules, bilayer scaffolds	Osteochondral repair, bone regeneration, wound healing	[224,225,227,241,242]
Recombinant human-like collagen (RHC)	E. coli; P. pastoris	Rat	Composite scaffolds, hydrogels, functionalized hydrogels	Bone tissue engineering, bioactive scaffolds, skin repair	[[213], [214], [215], [216], [217],238], [239,250]
Recombinant human type III collagen (rhCol III)	E. coli; P. pastoris; CHO	Rabbit; Rat	Photocrosslinked or composite hydrogels, powders, nanoparticle-loaded hydrogels	Wound healing, chronic ulcer repair, antimicrobial/antioxidant therapy	[223,231,233,234,236,237,244,246,249]
Recombinant human type I collagen (rhCol I)	P. pastoris; CHO; Plants	Rat	Hydrogels, crosslinked scaffolds, metal–polyphenol hydrogels, composites	Skin and soft tissue regeneration, multifunctional scaffolds	[230], [258],
Recombinant type III collagen (rCol III)	E. coli	Mouse	wound dressings, hydrogel	Acute and chronic wound repair	[228,262]
Recombinant human type II collagen (rhCII)	P. pastoris; CHO	Rabbit; miniature pig	Hydrogels, composite scaffolds with chondrocytes	Cartilage regeneration, joint repair	[220,221]
Recombinant humanized collagen (rHC)	E. coli; P. pastoris	Rat	Crosslinked hydrogels	Wound healing, tissue regeneration	[253]
Recombinant collagen (CF)	P. pastoris	Rabbit; Rat	3D-printed photocrosslinkable hydrogels, wound dressings	Bioprinted scaffolds, osteochondral repair	[226,230]

6. Regulatory policies and clinical translation

Recombinant collagen, owing to its excellent bioactivity, biocompatibility, and batch-to-batch consistency, is increasingly recognized as a next-generation alternative to animal-derived collagen. However, its translation from laboratory innovation to clinical reality involves a complex, multi-stage process encompassing molecular design, expression optimization, process scale-up, preclinical validation, regulatory approval, and clinical implementation (Fig. 11).

Fig. 11.

Roadmap for the clinical translation of recombinant collagen. The process typically involves four sequential stages: (1) Laboratory research and development, including plasmid construction, host optimization, structural characterization, and functional validation; (2) Pilot-scale studies, focusing on fermentation optimization, purification refinement, and preliminary validation of structure and function; (3) Large-scale production, involving high-density fermentation, establishment of GMP-compliant manufacturing platforms, and development of robust quality control systems; and (4) Clinical translation and regulatory approval, which encompasses preclinical and clinical safety evaluation, immunogenicity assessment, regulatory review, and final product approval.

At the research stage, critical tasks include rational sequence design, optimization of expression hosts, screening of production conditions, purification, and functional characterization to obtain recombinant collagens with authentic triple-helical structure and bioactivity. Process development and scale-up focus on fermentation optimization, purification efficiency, structural fidelity, batch consistency, and quality management, ensuring clinical-grade yield and reproducibility. Preclinical evaluation employs both small- and large-animal models to assess biocompatibility, immunogenicity, degradation kinetics, and regenerative efficacy, establishing a robust evidence base for clinical translation. In the translational and regulatory phase, compliance with Good Manufacturing Practice (GMP) and adherence to national or international regulatory frameworks are essential, encompassing raw material traceability, impurity control, and verification of structural and functional integrity. Ultimately, clinical trials and product registration with authorities such as the FDA, EMA, or NMPA are required to secure market approval.

6.1. International regulatory policies

As recombinant collagen advances toward clinical use, regulatory compliance has become a decisive determinant of translational success. While the regulatory systems of the United States, European Union, and China share fundamental principles of safety, efficacy, and traceability, they differ significantly in classification, approval pathways, and technical requirements. In the United States, the Food and Drug Administration (FDA) classifies collagen-based products by their intended use—as medical devices, biologics, or cosmetics—and evaluates them via the 510(k)/PMA or IND/BLA pathways. Regulatory assessments emphasize biological safety (ISO 10993 series), immunogenicity, degradation byproducts, and manufacturing consistency under current Good Manufacturing Practice (cGMP).

The European Union, under MDR 2017/745, mandates CE certification based on clinical evaluation, risk management, and lifecycle quality control. Biological safety verification aligns with ISO 10993 and must be supported by preclinical and clinical data. Post-market clinical follow-up and surveillance requirements are particularly stringent [275]. In China, the National Medical Products Administration (NMPA) has promulgated the China Drug Regulatory Science Action Plan (“Action Plan”), a strategic initiative intended to advance regulatory science research for medical products and to establish robust frameworks for evaluating their safety and efficacy [[276], [277], [278]]. Recombinant collagen-based products are generally categorized by the NMPA as Class II or Class III medical devices, which entails rigorous requirements including type testing, clinical evaluation, comprehensive risk assessment, and adherence to a full quality management system. Particular attention is paid to immunogenicity, degradation kinetics, and residual safety. While these three regulatory systems converge on rigorous safety and quality requirements, differences in classification, documentation, and approval timelines mean that early engagement with regulatory agencies is essential to streamline development, accelerate approval, and reduce commercialization risk.

6.2. Challenges in clinical translation

Despite its substantial therapeutic potential, recombinant collagen still faces significant barriers on the path to clinical application [279]. Its production is inherently complex and costly, requiring high-efficiency expression systems, correct protein folding, and precise post-translational modifications such as hydroxylation and glycosylation, making preparation far more demanding than that of animal-derived collagen. Achieving GMP-grade quality further necessitates stringent control over fermentation, purification, and endotoxin removal, particularly in prokaryotic systems. Moreover, yield and modification fidelity remain limiting factors, as eukaryotic hosts often exhibit low productivity and heterogeneous modifications, resulting in insufficient hydroxyproline content, local structural instability, and exposure of immunogenic epitopes that compromise performance in long-term implants and mechanically demanding environments. The regulatory landscape also poses challenges: despite several products having received CE or NMPA approval, dedicated frameworks tailored to recombinant collagen are still lacking, and products classified as novel devices or biologics must undergo extensive safety, immunogenicity, and efficacy assessments, prolonging and increasing the cost of registration. Finally, the industrial and commercial ecosystem remains underdeveloped; animal-derived collagen continues to dominate due to mature manufacturing infrastructure and lower production costs, while the high expense and price sensitivity of recombinant collagen products hinder their widespread adoption and large-scale commercialization.

Consequently, the paradox of “abundant laboratory research but limited marketed products” arises from interrelated constraints in production technology, molecular modification, regulatory frameworks, and industrial maturity. Future breakthroughs will depend on host engineering and metabolic regulation to enhance yield and fidelity; smart manufacturing to reduce cost and enable scale-up; unified quality and regulatory standards to accelerate approval; and collaborative demonstration projects between academia, industry, and healthcare to drive adoption. Through these synergistic strategies, recombinant collagen can be more effectively transformed from a laboratory innovation into a clinically and commercially viable platform, fulfilling its potential as a foundational biomaterial in regenerative medicine.

7. Conclusion and outlook

Recombinant collagen is rapidly emerging as a transformative class of biomaterials at the intersection of molecular engineering and regenerative medicine. By enabling precise control over amino acid sequences, supramolecular assembly, and functional motif integration, recombinant platforms have overcome many of the longstanding limitations of animal-derived and synthetic collagens, including immunogenicity, batch variability, and limited bioactivity. These advances have laid a strong foundation for the design of next-generation collagen-based materials that are safer, more functional, and more clinically relevant, driving their adoption in applications ranging from tissue scaffolds and wound dressings to injectable formulations and organ repair.

Despite this progress, significant challenges continue to hinder the clinical translation and large-scale deployment of recombinant collagen. Expression efficiency remains limited, incomplete post-translational modifications compromise structural fidelity, and standardized biomanufacturing pipelines are still lacking. Furthermore, regulatory frameworks specific to recombinant collagen-based devices and implants are evolving, necessitating comprehensive validation of structural integrity, bioactivity, immunogenicity, and long-term safety. The combination of complex production requirements, high manufacturing costs, and stringent regulatory expectations underscores the need for integrated solutions that bridge fundamental research with translational and industrial readiness.

Looking ahead, the field is poised to transition from biomimetic replication to programmable, intelligent, and clinically targeted applications. Future breakthroughs will likely arise from four converging directions: high-performance biomanufacturing, through synthetic biology, metabolic reprogramming, and CRISPR-enabled host engineering to enhance yield, modification precision, and batch consistency; AI-driven collagen design, leveraging deep learning and structural modeling to integrate bioactive motifs and accelerate the design–validation–application cycle; integration with advanced fabrication technologies, including 3D bioprinting, microfluidics, and molecular self-assembly, to construct spatially organized and dynamically responsive tissue architectures; and clinical-grade translation, through the establishment of GMP-compliant production processes, robust immunological evaluation systems, and application-specific performance standards. In this context, recombinant collagen is not merely a substitute for its native counterpart but a programmable and customizable biomaterial platform. Its continued evolution—powered by artificial intelligence, gene editing, and next-generation manufacturing—will enable its full clinical potential and usher in a new era of precision biomaterials and regenerative therapeutics.

CRediT authorship contribution statement

Huixia He: Writing – original draft, Visualization, Methodology, Investigation. Mingzhu Ye: Visualization, Investigation. Guoqi Cui: Visualization, Investigation. Jianxi Xiao: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.