Focal Adhesion of Collagen-Based Bone Grafting Materials Enhances Bone Regeneration

1. Introduction

Except in cases of bone defects caused by accidents, surgery, or injury, bone can repair itself by activating surrounding osteoprogenitor cells, leaving no residual scars. However, severe defects that are beyond bone’s self-healing capacity can lead to bone nonunion, callus formation, and even permanent bone loss. Bone grafts are often used for repairing such defects.

Autologous bone grafting is the gold standard for the repair of severe bone defects. Autologous grafts facilitate the repair or regeneration of bone tissue through the differentiation and proliferation of adjacent or transplanted osteoblasts. These grafts are associated with a low risk of rejection, and because of their low antigenicity, the grafts can promote bone regeneration [1]. However, autologous bone grafting has some major disadvantages, such as poor osseointegration with artificial joints or tooth implants. Excessive bone constriction may occur when the defect is >9 cm or when the surrounding tissues do not provide sufficient blood supply due to scarring, infection, or irradiation [2]. Moreover, autologous grafting involves the sacrifice of healthy bone tissue at the donor site, resulting in additional morbidity [3]. Harvesting bone from appropriate donor sites requires a complex learning curve and may cause postoperative complications.

In addition to autologous bone grafts, alternative grafts such as allogenous grafts, xenografts, and allografts are used for bone repair. These alternative grafts exhibit various physicochemical characteristics and varying reliability in surgical treatments [4,5]. Among all alternative grafts, collagen-based bone grafting materials are widely used in clinical settings. These materials are produced by combining collagen with other materials, thereby increasing bone repair and regeneration capacities.

Bone is a hard connective tissue with a hierarchical structure; it is composed of matrix, cells, and bioactive factors. The bone matrix is mainly composed of type I collagen and hydroxyapatite (HAP). Collagen fibers serve as a template for mineralization and play key roles in determining specific properties, such as compressive and shear behaviors, fracture mode, and toughness as well as reinforcing bone under external stress from applied loads [6,7].

Osteoblasts are anchorage-dependent cells. Therefore, the initial adhesion of osteoblasts to the biomaterial scaffold is pivotal for their migration, differentiation, and proliferation and subsequent bone formation. The adhesion of cells to the biomaterial surface is a complex process involving cell attachment, cell spreading, and cell–scaffold interaction; this process is called focal adhesion.

Collagen is the most abundant protein, constituting more than one-third (by weight) of the body’s total protein content [8]. Type I collagen is the most common type of collagen found in the extracellular matrix (ECM), particularly in bone tissue [9]. The ECM plays vital roles in the morphogenesis and cellular metabolism of new tissues, conferring mechanical and biochemical properties [10]. The collagen matrix is essential for bone tissue engineering [11] because of its abundance, biocompatibility, high porosity, ability to combine with other materials, easy processing, hydrophilicity, low antigenicity, and absorbability [12,13]. It can promote cell adhesion, differentiation, and proliferation.

Recently, scientists have designed innovative collagen-based biomaterial scaffolds, leveraging the extensive evidence on collagen organization, structure, and properties. The use of these materials has considerably enhanced bone engineering. The design of resorbable collagen-based medical scaffolds requires knowledge of the anatomy and biological function of tissues or organs as well as an understanding of the role of collagen’s physicochemical properties and structure in tissue or organ regeneration. Different collagen-based scaffolds have been developed for different bone engineering applications. These scaffolds promote biological responses, such as cell signaling, and serve as artificial biomimetic extracellular matrices that guide bone tissue regeneration. The initial step of bone regeneration involves the adhesion of cells to the collagen-based scaffold; the subsequent steps are material–tissue interaction, cell differentiation, cell proliferation, and new bone formation [14,15].

Cell adhesion is initially activated by the interaction of the collagen matrix with integrin. The mechanisms through which integrin signaling activates cell adhesion are discussed in the following sections.

2. Mechanisms of Collagen Binding and Cell Adhesion

Integrin, a cell surface receptor, plays an essential role in regulating cell signaling, migration, survival, and adhesion to various ECMs, including collagen, fibronectin (FN), and laminin (LN) [16,17,18]. The arginine–glycine–aspartic acid (Arg–Gly–Asp [RGD]) peptide of collagen constitutes a specific site for its recognition by integrin. Integrin is a key regulator of cell–cell and cell–extracellular microenvironment communication.

Various ECM-binding integrins that facilitate the adhesion of osteoblasts on biomaterial surfaces have been identified. Six integrins play major roles in cell adhesion: α1β1, α2β1, α3β1, αVβ1, α5β1, and α11β1 (Geographic abstract A) [16,19,20].

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is activated upon integrin binding to the ECM at the site of focal adhesion [21]. Integrin-mediated adhesion at tyrosine 397 leads to the autophosphorylation of FAK, creating a binding site for the proto-oncogene tyrosine-protein kinase (Src) homology 2 (SH2) domain of Src; the FAK–Src complex, in turn, phosphorylates other tyrosine residues in FAK, thus maximizing its kinase activity and creating additional protein-binding sites [22]. This active FAK–Src complex activates Ras-related C3 botulinum toxin substrate 1 (Rac1) by recruiting and phosphorylating the p130 Cas scaffold protein (p130 Crk-associated substrate, known as breast cancer anti-estrogen resistance 1 [Bcar1]) [23]. The phosphorylated p130 Cas recruits Dock 180 and engulfment and motility 1 (ELMO1; a Ced-12 ortholog) by binding to v-crk (sarcoma virus CT10 oncogene homolog crk; proto-oncogene, adaptor protein). The Dock 180-ELMO1 complex functions as an unconventional GEF for Rac1 and promotes the formation of membrane protrusions [24,25]. The activation of Rac1 and cell division cycle 42 (Cdc42) is also inhibited. This suppresses actomyosin contractility and enhances actin-mediated protrusion. The activity of Rac1 and Cdc decreases, whereas that of RhoA gradually increases, which promotes the formation of stress fibers and the maturation of focal adhesion (Geographic abstract C) [26,27].

The FAK–Src complex also phosphorylates paxillin-kinase linker (the GEF for Cdc and Rac1, which is also known as G-protein-coupled receptor kinase-interacting protein 2 [GIT2] and Pak-interacting exchange factor-beta [βPIX, also known as cloned out of library-1 {Cool-1} and Rho guanine nucleotide exchange factor 7 {Gef7}]). βPIX recruits and activates Rac1 through direct interactions with focal adhesions and membrane protrusions [28]. Notably, paxillin-kinase linker and βPIX are phosphorylated by Src, which further modulates their activity in response to integrin-mediated adhesion [29,30]. Thus, integrin signaling through Src family kinases (SFKs) can regulate the localization and activity of GEFs to control the formation of membrane protrusions.

The FAK–Src complex activates several pathways; subsequently, activated Rac and Cdc42 GTPases facilitate the formation of a membrane protrusion at the site of integrin binding in the early stages of cell spreading. Simultaneously, this complex, together with syndecans, mediates the suppression of actomyosin contractility by reducing the activity of RhoA. In the later stages of cell spreading, integrins promote the activity of several GEFs, leading to a shift in the balance between RhoA and Rac1 activity in favor of RhoA activity, thereby enhancing RhoA-mediated actomyosin contractility. The integrin α5β1 is particularly efficient in promoting the second phase of cell spreading, which may involve the force-induced activation of SFKs.

The enhanced activity of FAK promotes RhoA activity, thereby promoting cellular contractility through ROCK. Crosstalk between RhoA–ROCK and the extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway induces the phosphorylation of p44/42 MAPK (ERK1/ERK2) through MAPK kinase (MEK), which regulates the activity of the osteogenic transcription factor runt-related transcription factor 2 (RUNX2). RUNX2 controls the expression of osteogenic genes such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein, ultimately driving differentiation toward mature osteoblasts (Geographic abstract D) [31].

3. Collagen-Based Materials

Collagen-based materials promote the differentiation of human-induced pluripotent stem cells into osteoblasts [32]. The collagen matrix combined with other ECM or materials also enhances cell differentiation and proliferation. Other ECMs or materials include LN [33], FN [34,35], chitosan (CS) [36], HAP [37,38], calcium phosphate cement (TCP), fibrin, and fibrinogen [39]. Collagen-based materials are also applied as coating on the implant to enhance osteoblast adhesion to the implant surface and osseointegration between bone and the implant [40,41,42,43,44]. The techniques and mechanisms are described in the following sections.

3.1. Orientation of Collagen Fibers

The orientation of collagen-coated poly-lactide-co-glycolide (PLGA)/poly-caprolactone (PCL) fibers was reported to enhance bone regeneration through cell adhesion. HanBang Chen et al. demonstrated that adipose-derived stem cells exhibited the highest expression of adhesion-related genes, such as those encoding integrin β1, cadherin 11, Fn-1, LN, and N-cadherin [45]. Electrospinning technology was used to fabricate collagen/HAP fibers to improve bone regeneration. Collagen/HAP fibers support cell adhesion and bone regeneration. Yuanyuan Zhou et al. produced collagen/HAP composite fibers through electrospinning, and after 6 days of culture, the composite fibers exhibited higher viability and ALP activity than did myeloma cells [46].

3.2. LN

Selective cell retention has been widely used as a bone tissue engineering technique. LN is a main component of the ECM and the basement membrane. LN plays a key role in mediating cell–matrix adhesion, leading to cell proliferation and differentiation [32,33]. A collagen-binding domain (CBD) containing the core functional amino acid sequences of LN-4 (CBD-LN peptide) was introduced on the functional surface of a collagen-based decalcified bone matrix scaffold. The decalcified bone matrix/CBD-LN scaffold maintained osteoblast proliferation and induced osteogenic differentiation through early cell adhesion mediated by upregulated integrin β1 expression [33]. In tissue engineering, bioactive molecules have been introduced into three-dimensional porous scaffolds to mimic the in vivo microenvironment.

3.3. FN

FN is an ECM glycoprotein with a size ranging from 230 to 270 kDa. The dimer is formed by α and β subunits. FN includes types I, II, and II domains. The type III domain is the most sensitive to unfolding. Several receptors can bind to FN—for example, α2β1, α3β1, αvβ1, and α5β1—and improve focal adhesion, cell proliferation, and cell migration [47]. FN combined with type I collagen can promote focal adhesion, as evidenced by elevated filopodium formation, increased cell circularity, and accelerated spreading in mesenchymal stromal cell line [34]. Coating FN combined with OCN on the collagen matrices enhanced the adhesion of osteoblast-like cells (MC3T3-E1) and mRNA levels of osteogenic markers, such as RUNX2, ALP, and collagen type I in these cells [35].

3.4. Ceramic and Combined Materials

A hybrid scaffold composed of granular HAP and collagen was designed to mimic the microenvironment for the adhesion, viability, and osteoinduction of human bone marrow-derived mesenchymal stem cells (BMSCs) [48].

Moreau et al. reported that collagen incorporated into calcium phosphate bone cement increased the attachment and osteogenesis of osteoblasts [49]. Gutierrez et al. [50] proved the adhesion activity of HAP. The nano apatite–collagen composite appeared more similar to natural bone in terms of biomimetics than was nano apatite cement without collagen [49]. Functionally graded CO₃ apatite–collagen containing magnesium (FGMg-Ap-collagen) was also reported to enhance osteoblast adhesion to apatite and to promote bone formation [51]. CS combined with collagen/HAP in mesenchymal stem cells (MSCs) [52] and rats [53] and collagen/β-TCP in the human osteoblast cell line MG63 [54] increased cell adhesion and proliferation, resulting in osteogenesis.

Yu et al. reported that intrafibrillar mineralized Col-HA-based scaffolds, constructed with either cellular or lamellar microstructures, exhibited enhanced bone regeneration capacity in a mouse model. Moreover, Fe/Mn incorporation promoted the osteogenic potential of the lamellar scaffolds, facilitating the in vitro osteogenic differentiation of BMSCs and the in vivo bone regeneration in the presence of fresh bone marrow cells [55].

3.5. CS

CS, a nontoxic natural polymer, is primarily composed of −1,4-linked N-acetyl-D-glucosamine and D-glucosamine units. CS exhibits high solubility in dilute acidic solution with pH < 6.5 [56]. This polymer forms covalent bonds with FN, improving cell adhesion [57]. CS binding to collagen/HAP or CS/collagen/β-TCP leads to the formation of a three-dimensional structure that enhances cell adhesion and proliferation, resulting in osteogenesis [53,54]. Osteoblast adhesion increases with increasing β-TCP and CS contents [54,58].

3.6. Fibrin and Fibrinogen

Fibrin is widely used for enhancing the focal adhesion of collagen [59]. Santos et al. reported that the combination of collagen sponge and fibrin glue exerted hemostatic effects and ensured more favorable bone formation than did only collagen sponge [59,60]. B.-S. Kim et al. found that type I collagen-derived atelocollagen/fibrin composite gel with the addition of an optimal concentration of fibrinogen supported human MSC growth in vitro and bone formation in vivo [61].

3.7. Cytokines and Chemokines

Cytokines or chemokines, such as bone morphogenetic proteins (BMPs), chemotactic cytokine ligand (CXCL)12, and CXCL13, can enhance cell adhesion to the scaffold. Osteoblast adhesion to collagen or collagen-based materials leads to increased cell proliferation. BMP4 was immobilized in a CBD and bound to the collagen–polyglycolic hybrid scaffold; the BMP-immobilized hybrid scaffold supported the adhesion and proliferation of MSCs [62].

Claude Laflamme et al. revealed that a mixture of BMP-2/BMP-7 homodimers enhanced osteoblast adhesion and growth following culture on a collagen scaffold. Osteoblast adhesion and proliferation increased 4 days after culture on the collagen scaffold. BMP-2/BMP-7 promoted bone regeneration through different mechanisms involving interleukin-6 and matrix metalloproteinase inhibitors [63].

Sylvia Weeks et al. reported that the incorporation of the chemokines CXCL12 and CXCL13 into a poly-L-lactic acid–collagen-based scaffold increased cell adhesion. Combining CXCL12 with an FN- and collagen-coated scaffold increased MSC adhesion to the scaffold through α4 and α5β1 binding [64]. This scaffold promoted the differentiation of MSCs into osteoblasts, resulting in bone formation.

3.8. Small Molecules

Grafting collagen with other material to fabricate a scaffold is an effective strategy for enhancing cell proliferation and ALP expression [65]. Collagen can improve cell adhesion because of the presence of its Pro-α3(V) chain in bone. Through the N-terminal peptide of this chain, collagen adheres to osteosarcoma cells [66]. OCN, a small bone ECM protein, accelerated bone formation in a rat model when added to HAP/collagen composites. Histological findings indicate that OCN activates both osteoblasts and osteoclasts during early bone formation [37].

3.9. Implants

Collagen is widely used to coat biomedical implants. Geißler and Hempel et al. have reported that the collagen type I coating of Ti6Al4V promoted the initial adhesion of osteoblasts in the presence of fetal calf serum. Moreover, 60% to 90% of all osteoblasts adhered to collagen type I–coated surfaces, whereas 30% of the initial cell number remained adhered to uncoated surfaces. Collagen type I, which includes the RGD peptide, effectively promotes cell adhesion by interacting with α1β1 and α2β1 [40]. The type I collagen–coated Ti–6Al–4V alloy facilitated osseointegration and bone-to-implant contact [41].

Titanium-based implants exhibit osseointegration and are widely used in dental and orthopedic care. However, cell growth and differentiation capacity on the surface of titanium-based implants are limited. To address these limitations, various functionalization strategies for titanium surfaces have been developed—for example, bioactivated coating. One of the most used peptides to functionalize biomaterials is a cell adhesion peptide containing the RGD sequence, which is found in collagen, FN, and bone sialoprotein. RGD peptide–enriched materials interact with integrin on the cell surface and can enhance the proliferation of osteoblasts and bone marrow-derived stem cells [42].

In dental care, zirconia is used to manufacture implants. To enhance implant compatibility, nano-HAP is bound to the surface first and then type I collagen is immobilized on the surface. Compared with cases with no coating or with nano-HAP-only coating, cases with nano-HAP-collagen coating exhibited increased osteoblast attachment and spreading on the surface; higher osteoblast differentiation was confirmed by higher ALP activity and mineralization [43]. HAP and extensive collagen coating of an implant surface can facilitate cell attachment because of the presence of increased hydroxyl groups on the surface, which results in the formation of a low contact angle and the activation of carboxylic groups, which are beneficial for osteoblast adhesion and proliferation [44]. A list of articles reporting the modification of cell adhesion by collagen-based bone grafting materials for enhancing bone regeneration is provided in Table 1.

4. Conclusions

Over the last decades, extensive research has been conducted on collagen-based materials to improve their biological and mechanical properties for supporting efficient bone regeneration. Advanced strategies for regulating collagen’s structure and properties by modifying materials and refining processing techniques can yield highly biomimetic substrates, which can advance tissue engineering.

Collagen-based materials enhance FAK activity and activate the ERK–MAPK pathway. This stimulates the phosphorylation of ERK1/ERK2 through MEK, regulating the activity of osteogenic transcription factor RUNX2. RUNX2 controls the expression of osteogenic genes such as OCN, ALP, and bone sialoprotein, ultimately driving differentiation toward mature osteoblasts.