Affinity For Collagen Research Articles

Reversibly photoswitchable protein assemblies with collagen affinity for in vivo photoacoustic imaging of tumors.

Recent advancements in photoacoustic (PA) imaging have leveraged reversibly photoswitchable chromophores, known for their dual absorbance states, to enhance imaging sensitivity through differential techniques. Yet, their deployment in tumor imaging has faced obstacles in achieving targeted delivery with high efficiency and specificity. Addressing this challenge, we introduce innovative protein assemblies, DrBphP-CBD, by genetically fusing a photosensory module from Deinococcus radiodurans bacterial phytochrome (DrBphP) with a collagen-binding domain (CBD). These protein assemblies form sub-100-nanometer structures composed of 24 DrBphP dimers and 12 CBD trimers, presenting 24 protein subunits. Their affinity for collagens, combined with impressive photoswitching contrast, markedly improves PA imaging precision. In various tumor models, intravenous administration of DrBphP-CBD has demonstrated enhanced tumor targeting and retention, augmenting contrast in PA imaging by minimizing background noise. This strategy underscores the clinical potential of DrBphP-CBD as PA contrast agents, propelling photoswitchable chromoproteins to the forefront of precise cancer diagnosis.

The surface charge of electroactive materials governs cell behaviour through its effect on protein deposition

The precise mechanisms underlying the cellular response to static electric cues remain unclear, limiting the design and development of biomaterials that utilize this parameter to enhance specific biological behaviours. To gather information on this matter we have explored the interaction of collagen type-I, the most abundant mammalian extracellular protein, with poly(vinylidene fluoride) (PVDF), an electroactive polymer with great potential for tissue engineering applications. Our results reveal significant differences in collagen affinity, conformation, and interaction strength depending on the electric charge of the PVDF surface, which subsequently affects the behaviour of mesenchymal stem cells seeded on them. These findings highlight the importance of surface charge in the establishment of the material–protein interface and ultimately in the biological response to the material. Statement of significanceThe development of new tissue engineering strategies relies heavily on the understanding of how biomaterials interact with biological tissues. Although several factors drive this process and their driving principles have been identified, the relevance and mechanism by which the surface potential influences cell behaviour is still unknown. In our study, we investigate the interaction between collagen, the most abundant component of the extracellular matrix, and poly(vinylidene fluoride) with varying surface charges. Our findings reveal substantial variations in the binding forces, structure and adhesion of collagen on the different surfaces, which collectively explain the differential cellular responses. By exposing these differences, our research fills a critical knowledge gap and paves the way for innovations in material design for advanced tissue regeneration strategies.

Optimized Allogenic Decellularized Meniscal Scaffold Modified by Collagen Affinity Stromal Cell–Derived Factor SDF1α for Meniscal Regeneration: A 6- and 12-Week Animal Study in a Rabbit Model

Background: Total meniscectomy for treating massive meniscal tears may lead to joint instability, cartilage degeneration, and even progressive osteoarthritis. The meniscal substitution strategies for advancing reconstruction of the meniscus deserve further investigation. Hypothesis: A decellularized meniscal scaffold (DMS) modified with collagen affinity stromal cell–derived factor (C-SDF1α) may facilitate meniscal regeneration and protect cartilage from abrasion. Study Design: Controlled laboratory study. Methods: The authors first modified DMS with C-SDF1α to fabricate a new meniscal graft (DMS-CBD [collagen-binding domain]). Second, they performed in vitro studies to evaluate the release dynamics, biocompatibility, and differentiation inducibility (osteogenic, chondrogenic, and tenogenic differentiation) on human bone marrow mesenchymal stem cells. Using in vivo studies, they subjected rabbits that received medial meniscectomy to a transplantation procedure to implement their meniscal graft. At postoperative weeks 6 and 12, the meniscal regeneration outcomes and chondroprotective efficacy of the new meniscal graft were evaluated by macroscopic observation, histology, micromechanics, and immunohistochemistry tests. Results: In in vitro studies, the optimized DMS-CBD graft showed notable biocompatibility, releasing efficiency, and chondrogenic inducibility. In in vivo studies, the implanted DMS-CBD graft after total meniscectomy promoted the migration of cells and extracellular matrix deposition in transplantation and further facilitated meniscal regeneration and protected articular cartilage from degeneration. Conclusion: The new meniscal graft (DMS-CBD) accelerated extracellular matrix deposition and meniscal regeneration and protected articular cartilage from degeneration. Clinical Relevance: The results demonstrate that the DMS-CBD graft can serve as a potential meniscal substitution after meniscectomy.

An engineered immunocytokine with collagen affinity improves the tumor bioavailability, tolerability, and therapeutic efficacy of IL-2

The clinical utility of human interleukin-2 (hIL-2) is limited by its short serum half-life, preferential activation of regulatory T (TReg) over immune effector cells, and dose-limiting toxicities. We previously engineered F10 immunocytokine (IC), an intramolecularly assembled cytokine/antibody fusion protein that linked hIL-2 to an anti-IL-2 antibody (denoted F10) that extended IL-2 half-life and augmented the immune effector to TReg ratio. Here, we leveraged molecular engineering to improve the anti-tumor therapeutic efficacy and tolerability of F10 IC by developing an iteration, denoted F10 IC-CBD (collagen binding domain), designed for intratumoral administration and in situ retention based on collagen affinity. F10 IC-CBD retained IL-2 bioactivity exclusively in the tumor and eliminated IL-2-associated toxicities. Furthermore, F10 IC exhibited potent single-agent therapeutic efficacy and synergy with systemic immune checkpoint blockade and elicited an abscopal response in mouse tumors models. This engineered fusion protein presents a prototype for the design of intratumoral therapies.

Genetically encoded zinc-binding collagen-like protein hybrid hydrogels for wound repair

Incorporating zinc oxide nanoparticles (ZnOnps) into collagen is a promising strategy for fabricating biomaterials with excellent antibacterial activity, but modifications are necessary due to the low zinc binding affinity of native collagen, which can cause disturbances to the functions of both ZnOnps and collagen and result in heterogeneous effects. To address this issue, we have developed a genetically encoded zinc-binding collagen-like protein, Zn-eCLP3, which was genetically modified by Scl2 collagen-like protein. Our study found that Zn-eCLP3 has a binding affinity for zinc that is 3-fold higher than that of commercialized type I collagen, as determined by isothermal titration calorimetry (ITC). Using ZnOnps-coordinated Zn-eCLP3 protein and xanthan gum, we prepared a hydrogel that showed significantly stronger antibacterial activity compared to a collagen hydrogel prepared in the same manner. In vitro cytocompatibility tests were conducted to assess the potential of the Zn-eCLP3 hydrogel for wound repair applications. In vivo experiments, which involved an S. aureus-infected mouse trauma model, showed that the application of the Zn-eCLP3 hydrogel resulted in rapid wound regeneration and increased expression of collagen-1α and cytokeratin-14. Our study highlights the potential of Zn-eCLP3 and the hybrid hydrogel for further studies and applications in wound repair.

A collagen-binding SIRPαFc fusion protein for targeted cancer immunotherapy

Collagen is abundant but exposed in tumor due to the abnormal tumor blood vessels, thus is considered as a tumor-specific target. The A3 domain of von Willebrand factor (vWF A3) is a kind of collagen-binding domain (CBD) which could bind collagen specifically. Previously we reported a chemosynthetic CBD-SIRPαFc conjugate, which could block CD47 and derived tumor-targeting ability by CBD. CBD-SIRPαFc conjugate represented improved anti-tumor efficacy with increased MHC II+ M1 macrophages, but the uncertain coupling ratio remained a problem. Herein, we produced a vWF A3-SIRPαFc fusion protein through eukaryotic expression system. It was examined at both molecular and cellular levels with its collagen affinity, uninfluenced original affinity to targets and phagocytosis-promoting function compared to unmodified SIRPαFc. Living imaging showed that vWF A3-SIRPαFc fusion protein derived the improved accumulation and retention in tumor than SIRPαFc. In the MC38 allograft model, vWF A3-SIRPαFc demonstrated a superior tumor-suppressing effect, characterized by increased MHC II+ M1 macrophages and T cells (particularly CD4+ T cells). These results revealed that vWF A3-SIRPαFc fusion protein derived tumor-targeting ability, leading to improved anti-tumor immunotherapeutic efficacy compared to SIRPαFc. Altogether, vWF A3 improved the anti-tumor efficacy and immune-activating function of SIRPαFc, supporting targeting tumor collagen as a possible targeted strategy.

Characterization of LL37 Binding to Collagen through Peptide Modification with a Collagen-Binding Domain.

Collagen-based biomaterials loaded with antimicrobial peptides (AMPs) present a promising approach for promoting wound healing while providing protection against infections. In our previous work, we modified the AMP LL37 by incorporating a collagen-binding domain (cCBD) as an anchoring unit for collagen-based wound dressings. We demonstrated that cCBD-modified LL37 (cCBD-LL37) exhibited improved retention on collagen after washing with PBS. However, the binding mechanism of cCBD-LL37 to collagen remained to be elucidated. In this study, we found that cCBD-LL37 showed a slightly higher affinity for collagen compared to LL37. Our results indicated that cCBD inhibited cCBD-LL37 binding to collagen but did not fully eliminate the binding. This suggests that cCBD-LL37 binding to collagen may involve more than just one-site-specific binding through the collagen-binding domain, with non-specific interactions also playing a role. Electrostatic studies revealed that both LL37 and cCBD-LL37 interact with collagen via long-range electrostatic forces, initiating low-affinity binding that transitions to close-range or hydrophobic interactions. Circular dichroism analysis showed that cCBD-LL37 exhibited enhanced structural stability compared to LL37 under varying ionic strengths and pH conditions, implying potential improvements in antimicrobial activity. Moreover, we demonstrated that the release of LL37 and cCBD-LL37 into the surrounding medium was influenced by the electrostatic environment, but cCBD could enhance the retention of peptide on collagen scaffolds. Collectively, these results provide important insights into cCBD-modified AMP-binding mechanisms and suggest that the addition of cCBD may enhance peptide structural stability and retention under varying electrostatic conditions.

Formation of DNA nanotubes increases uptake into fibroblasts via enhanced affinity for collagen

DNA nanostructures are promising delivery carriers because of their flexible structural design and high biocompatibility. Selectivity in cellular uptake is an important factor in the development of DNA-nanostructure-based delivery carriers. In this study, DNA nanotubes were selected as the DNA structures, and their selectivity for cellular uptake and the mechanisms involved were investigated. Unlike DNA nanostructures such as polypod-like nanostructured DNA or DNA tetrahedrons, which are easily taken up by macrophages, the formation of DNA nanotubes increases uptake by fibroblasts and fibroblast-like cells. We focused on the collagen expressed in cells as a factor in this process, and found DNA nanotube formation increased the affinity for type I collagen compared with that of single-stranded DNA. Collagenase treatment removes collagen from fibroblasts and reduces the uptake of DNA nanotubes by fibroblasts. We directly observed DNA nanotube uptake by fibroblasts using transmission electron microscopy, whereby the nanotubes were distributed on the cell surface, folded, fragmented, and taken up by phagocytosis. In conclusion, we demonstrated a novel finding that DNA nanotubes are readily taken up by fibroblasts and myoblasts.

Role of heat shock protein 47 in platelet glycoprotein VI dimerization and signaling

BackgroundHeat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL. ObjectivesIn this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling. MethodsHSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling. ResultsUsing subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2β1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling. ConclusionThe present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation.

Valorization of Salmo salar Skin Waste for the Synthesis of Angiotensin Converting Enzyme-1 (ACE1) Inhibitory Peptides.

One of potential inhibitors which is widely used for the clinical treatment of COVID-19 in comorbid patients is Angiostensin Converting Enzyme-1 (ACE1) inhibitor. A safer peptide-based ACE1 inhibitor derived from salmon skin collagen, that is considered as the by-product of the fish processing industry have been investigated in this study. The inhibitory activity against ACE1 was examined using in vitro and in silico methods. In vitro analysis includes the extraction of acid-soluble collagen, characterization using FTIR, Raman, UV–Vis, XRD, cytotoxicity assay, and determination of inhibition against ACE1. In silico method visualizes binding affinity, molecular interaction, and inhibition type of intact collagen and active peptides derived from collagen against ACE1 using molecular docking. The results of FTIR spectra detected amide functional groups (A, B, I, II, III) and imine proline/hydroxyproline, while the results of Raman displayed peak absorption of amide I, amide III, proline/hydroxyproline ring, phenylalanine, and protein backbone. Furthermore, UV–Vis spectra showed typical collagen absorption at 230 nm and based on XRD data, the chain types in the samples were α-helix. ACE1 inhibition activity was obtained in a concentration-dependent manner where the highest was 82.83% and 85.84% at concentrations of 1000, and 2000 µg/mL, respectively, and showed very low cytotoxicity at the concentration less than 1000 µg/mL. In silico study showed an interaction between ACE1 and collagen outside the active site with the affinity of − 213.89 kcal/mol. Furthermore, the active peptides of collagen displayed greater affinity compared to lisinopril, namely HF (His-Phe), WYT (Trp-Tyr-Thr), and WF (Trp-Phe) of − 11.52; − 10.22; − 9.58 kcal/mol, respectively. The salmon skin-derived collagen demonstrated ACE1 inhibition activity with a non-competitive inhibition mechanism. In contrast, the active peptides were predicted as potent competitive inhibitors against ACE1. This study indicated that valorization of fish by-product can lead to the production of a promising bioactive compound to treat COVID-19 patient with diabetic comorbid.

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

Collagen Type II—Chitosan Interactions as Dependent on Hydroxylation and Acetylation Inferred from Molecular Dynamics Simulations

Chitosan-collagen blends have been widely applied in tissue engineering, joints diseases treatment, and many other biomedical fields. Understanding the affinity between chitosan and collagen type II is particularly relevant in the context of mechanical properties modulation, which is closely associated with designing biomaterials suitable for cartilage and synovial fluid regeneration. However, many structural features influence chitosan's affinity for collagen. One of the most important ones is the deacetylation degree (DD) in chitosan and the hydroxylation degree (HD) of proline (PRO) moieties in collagen. In this paper, combinations of both factors were analyzed using a very efficient molecular dynamics approach. It was found that DD and HD modifications significantly affect the structural features of the complex related to considered types of interactions, namely hydrogen bonds, hydrophobic, and ionic contacts. In the case of hydrogen bonds both direct and indirect (water bridges) contacts were examined. In case of the most collagen analogues, a very good correlation between binding free energy and DD was observed.

In silico design of fusion keratinocyte growth factor containing collagen-binding domain for tissue engineering application

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies.

Enhancement of nucleus pulposus repair by glycoengineered adipose-derived mesenchymal cells

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates for repairing degenerated intervertebral discs through multiple means, including: i. Secretion of bioactive factors to regulate inflammation and, ii. The potential to differentiate into nucleus pulposus (NP)-like cells, which can integrate into host tissues. However, the differentiation ability of ADSCs to NP-like cells is limited, which emphasizes on the need for alternative approaches to regulate cell differentiations. Given that cell functions are influenced by interactions between the extracellular matrix (ECM) and cells, we hypothesize that cell surface modification promotes ADSCs adhesion and differentiation towards NP-like cells. In this study, cell surfaces of ADSCs were functionalized with unnatural sialic acid via metabolic glycoengineering. Subsequently, adhesion abilities of modified cells to three main ECM (laminin, collagen and fibronectin) were compared. The adhesion assay revealed that glycoengineered ADSCs had the highest affinity for collagen, compared to laminin and fibronectin. Moreover, cultures with collagen coated plates enhanced the differentiation of glycoengineered ADSCs to NP-like cells. Metabolic glycoengineering prolonged ADSCs viability. The glycoengineered ADSCs increased the height and elasticity of intervertebral discs, as well as the water content and ECM volumes of nucleus pulposus. In conclusion, metabolic glycoengineering of cell surfaces has a significant role in modulating cell biological functions and promoting NP tissue repair.

Effect of Chitosan Deacetylation on Its Affinity to Type III Collagen: A Molecular Dynamics Study.

The ability to form strong intermolecular interactions by linear glucosamine polysaccharides with collagen is strictly related to their nonlinear dynamic behavior and hence bio-lubricating features. Type III collagen plays a crucial role in tissue regeneration, and its presence in the articular cartilage affects its bio-technical features. In this study, the molecular dynamics methodology was applied to evaluate the effect of deacetylation degree on the chitosan affinity to type III collagen. The computational procedure employed docking and geometry optimizations of different chitosan structures characterized by randomly distributed deacetylated groups. The eight different degrees of deacetylation from 12.5% to 100% were taken into account. We found an increasing linear trend (R2 = 0.97) between deacetylation degree and the collagen–chitosan interaction energy. This can be explained by replacing weak hydrophobic contacts with more stable hydrogen bonds involving amino groups in N-deacetylated chitosan moieties. In this study, the properties of chitosan were compared with hyaluronic acid, which is a natural component of synovial fluid and cartilage. As we found, when the degree of deacetylation of chitosan was greater than 0.4, it exhibited a higher affinity for collagen than in the case of hyaluronic acid.

Collagen Fragments Produced in Cancer Mediate T Cell Suppression Through Leukocyte-Associated Immunoglobulin-Like Receptor 1.

The tumor microenvironment (TME) is a complex structure comprised of tumor, immune and stromal cells, vasculature, and extracellular matrix (ECM). During tumor development, ECM homeostasis is dysregulated. Collagen remodeling by matrix metalloproteinases (MMPs) generates specific collagen fragments, that can be detected in the circulation of cancer patients and correlate with poor disease outcome. Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is an inhibitory collagen receptor expressed on immune cells in the TME and in the circulation. We hypothesized that in addition to ECM collagen, collagen fragments produced in cancer can mediate T cell immunosuppression through LAIR-1. Our analyses of TCGA datasets show that cancer patients with high tumor mRNA expression of MMPs, collagen I and LAIR-1 have worse overall survival. We show that in vitro generated MMP1 or MMP9 collagen I fragments bind to and trigger LAIR-1. Importantly, LAIR-1 triggering by collagen I fragments inhibits CD3 signaling and IFN-γ secretion in a T cell line. LAIR-2 is a soluble homologue of LAIR-1 with higher affinity for collagen and thereby acts as a decoy receptor. Fc fusion proteins of LAIR-2 have potential as cancer immunotherapeutic agents and are currently being tested in clinical trials. We demonstrate that collagen fragment-induced inhibition of T cell function could be reversed by LAIR-2 fusion proteins. Overall, we show that collagen fragments produced in cancer can mediate T cell suppression through LAIR-1, potentially contributing to systemic immune suppression. Blocking the interaction of LAIR-1 with collagen fragments could be an added benefit of LAIR-1-directed immunotherapy.

Tendon-inspired fibers from liquid crystalline collagen as the pre-oriented bioink

Nature provides rich bionic resources for the construction of advanced materials with excellent mechanical properties. In this work, inspired by animal tendons, a bionic collagen fiber was developed using collagen liquid crystals as the pre-oriented bioink. The texture of liquid crystalline collagen observed from polarized optical microscopy (POM) showed the specific molecular pre-orientation. Meanwhile, the collagen spinning liquids exhibited a minimal rise in viscosity upon increasing concentration from 60 to 120 mg/mL, indicating the feasible processability. The collagen fiber, which was prepared via wet spinning without being denatured, exhibited the favorable orientation of fibrils along its axis as observed with FESEM and AFM. Thanks to the synergistic effects between pre-orientation and shearing orientation, the maximum tensile strength and Young's modulus of collagen fibers reached 9.98 cN/tex (219.29 ± 22.92 MPa) and 43.95 ± 1.11 cN/tex (966.20 ± 24.30 MPa), respectively, which were also analogous to those of tendon. In addition, the collagen fiber possessed a desirable wet strength. Benefiting from the natural tissue affinity of collagen, the as-prepared bionic collagen fiber possessed excellent wound suture performance and biodegradability in vivo, which offers a new perspective for the potential of widespread applications of collagen fibers in biomedical fields.

Engineered bridge protein with dual affinity for bone morphogenetic protein-2 and collagen enhances bone regeneration for spinal fusion.

The revolutionizing efficacy of recombinant human bone morphogenetic protein (rhBMP-2) for clinical spinal fusion is hindered by safety issues associated with the high dose required. However, it continues to be widely used, for example, in InFUSE Bone Graft (Medtronic). Here, we developed a translational protein engineering-based approach to reduce the dose and thereby improve the safety of rhBMP-2 delivered in a collagen sponge, as in InFUSE Bone Graft. We engineered a bridge protein with high affinity for rhBMP-2 and collagen that can be simply added to the product's formulation, demonstrating improved efficacy at low dose of rhBMP-2 in two mouse models of bone regeneration, including a newly developed spinal fusion model. Moreover, the bridge protein can control the retention of rhBMP-2 from endogenous collagenous extracellular matrix of tissue. Our approach may be generalizable to other growth factors and collagen-based materials, for use in many other applications in regenerative medicine.

Fabrication of Three-Dimensional Bioactive Composite Scaffolds for Hemostasis and Wound Healing.

Fabrication of 3D composite scaffolds was carried out by lyophilization of variable concentrations of collagen and chitosan gel solutions. Fibrinogen and thrombin aerosol were deposited over the surface of scaffolds to enhance hemostasis and wound healing. Composite scaffolds were characterized using differential scanning calorimetry, thermogravimetric analysis, and Fourier-transform infrared spectrophotometer to ascertain the aerosol deposition and stability. Scanning electron microscope showed multilayered porosity with pore size of ~30 μm and mushroom-like fibril growth of aerosol. A detailed investigation by surface plasmon resonance confirmed higher binding affinity of collagen toward the human blood platelets and erythrocytes in comparison to chitosan and was found to increase with the increase in blood cell concentration from 480.8 to 886.4 RU for erythrocytes. Scaffolds showed higher binding response for platelets than erythrocytes, while fibrinogen and thrombin showed no or limited interaction. Highest blood sorption of 83 ± 4% was observed in case of aerosol deposited scaffolds. Aerosol deposited scaffolds showed minimum clotting time of 20 ± 3 s and bleeding time of 38 ± 4 s, which was significantly lower compared to the scaffolds without aerosol treatment. Aerosol deposited composite scaffolds with 2:1 concentration of chitosan/collagen showed complete wound contraction by day 14, while 50% was observed in case of the control group. In vivo studies revealed that chitosan had a crucial role in the inflammatory phase, while collagen played an important role in the proliferation and maturation phase. The present study suggests that the fabricated 3D composite scaffolds with bioactive moieties may be a potential candidate for enhanced hemostasis and wound healing applications.

Targeting Activated Hepatic Stellate Cells Using Collagen-Binding Chitosan Nanoparticles for siRNA Delivery to Fibrotic Livers.

Activated hepatic stellate cells (aHSCs) are the main orchestrators of the fibrotic cascade in inflamed livers, with transforming growth factor-beta (TGF-β) being the most potent pro-fibrotic cytokine. Hence, aHSCs serve as interesting therapeutic targets. However, drug delivery to aHSCs is hindered by excessive collagen deposition in the extracellular matrix (ECM) and capillarization of liver sinusoids. Chitosan-nanoparticles (CS-NPs) show intrinsic affinity for collagen, holding potential for drug delivery to fibrotic livers. Here, we employed CS-NPs for anti-TGF-β siRNA delivery, promoting delivery into aHSCs via modification with platelet-derived growth factor receptor-beta binding peptides. In-vitro experiments using aHSCs demonstrated the association of unmodified CS-NPs to the collagen-rich ECM, with reduced intracellular accumulation. Peptide-modified CS-NPs showed a higher propensity to localize intracellularly; however, this was only the case upon ECM-collagen reduction via collagenase treatment. Peptide-modified CS-NPs were more potent than unmodified CS-NPs in reducing TGF-β expression, implying that while collagen binding promotes liver accumulation, it hinders cell-specific siRNA delivery. In-vivo, CS-NPs successfully accumulated in fibrotic livers via collagen binding. Similar to in-vitro findings, when mice were pretreated with collagenase-loaded CS-NPs, the accumulation of peptide-modified NPs increased. Our findings demonstrate the usefulness of NPs modification with targeting ligands and collagenase treatment for aHSCs targeting and highlight the importance of chitosan–collagen binding in drug delivery to fibrotic diseases.