Evaluating the Efficacy of a Thermoresponsive Hydrogel for Delivering Anti-Collagen Antibodies to Reduce Posttraumatic Scarring in Orthopedic Tissues.

Excessive posttraumatic scarring in orthopedic tissues, such as joint capsules, ligaments, tendons, muscles, and peripheral nerves, presents a significant medical problem, resulting in pain, restricted joint mobility, and impaired musculoskeletal function. Current treatments for excessive scarring are often ineffective and require the surgical removal of fibrotic tissue, which can aggravate the problem. The primary component of orthopedic scars is collagen I-rich fibrils. Our research team has developed a monoclonal anti-collagen antibody (ACA) that alleviates posttraumatic scarring by inhibiting collagen fibril formation. We previously established the safety and efficacy of ACA in a rabbit-based arthrofibrosis model. In this study, we evaluate the utility of a well-characterized thermoresponsive hydrogel (THG) as a delivery vehicle for ACA to injury sites. Crucial components of the hydrogel included N-isopropylacrylamide, poly(ethylene glycol) diacrylate, and hyaluronic acid. Our investigation focused on in vitro ACA release kinetics, stability, and activity. Additionally, we examined the antigen-binding characteristics of ACA post-release from the THG in an in vivo context. Our preliminary findings suggest that the THG construct exhibits promise as a delivery platform for antibody-based therapeutics to reduce excessive scarring in orthopedic tissues.

Corneal Wound Healing in the Presence of Antifibrotic Antibody Targeting Collagen Fibrillogenesis: A Pilot Study.

Highly organized collagen fibrils interlacing with proteoglycans form the crucial architecture of the cornea and facilitate its transparency. Corneal scarring from accidental injury, surgery, or infection alters this highly organized tissue, causing severe consequences, including blindness. There are no pharmacological or surgical methods to effectively and safely treat excessive corneal scarring. Thus, we tested the anticorneal scarring utility of a rationally designed anticollagen antibody (ACA) whose antifibrotic effects have already been demonstrated in nonocular models. Utilizing a rabbit model with an incisional corneal wound, we analyzed ACA's effects on forming collagen and proteoglycan-rich extracellular matrices in scar neotissue. We used microscopic and spectroscopic techniques to quantify these components and measure crucial parameters characterizing the structure and organization of collagen fibrils. Moreover, we analyzed the spatial distribution of collagen and proteoglycans in normal and healing corneas. Our study demonstrated significant changes in the quality and quantity of the analyzed molecules synthesized in scar neotissue. It showed that these changes extend beyond incision margins. It also showed ACA's positive impact on some crucial parameters defining proper cornea structure. This pilot study provides a stepping stone for future tests of therapeutic approaches that target corneal extracellular scar matrix assembly.

Extracellular Targets to Reduce Excessive Scarring in Response to Tissue Injury.

Excessive scar formation is a hallmark of localized and systemic fibrotic disorders. Despite extensive studies to define valid anti-fibrotic targets and develop effective therapeutics, progressive fibrosis remains a significant medical problem. Regardless of the injury type or location of wounded tissue, excessive production and accumulation of collagen-rich extracellular matrix is the common denominator of all fibrotic disorders. A long-standing dogma was that anti-fibrotic approaches should focus on overall intracellular processes that drive fibrotic scarring. Because of the poor outcomes of these approaches, scientific efforts now focus on regulating the extracellular components of fibrotic tissues. Crucial extracellular players include cellular receptors of matrix components, macromolecules that form the matrix architecture, auxiliary proteins that facilitate the formation of stiff scar tissue, matricellular proteins, and extracellular vesicles that modulate matrix homeostasis. This review summarizes studies targeting the extracellular aspects of fibrotic tissue synthesis, presents the rationale for these studies, and discusses the progress and limitations of current extracellular approaches to limit fibrotic healing.

Scar formation in the presence of mitomycin C and the anti-fibrotic antibody in a rabbit model of glaucoma microsurgery: A pilot study.

Purpose: This study aimed to evaluate the utility of a rationally engineered antibody that directly blocks collagen fibrillogenesis to reduce scar tissue formation associated with subconjunctival glaucoma surgery. Material and methods: Fourteen eyes of 7 adult rabbits underwent glaucoma filtering surgery using XEN 45 Gel Stent. The rabbits' eyes were divided randomly into three treatment groups: (i) treated with the antibody, (ii) treated with mitomycin C, and (iii) treated with the antibody and mitomycin C. Following surgeries, the intraocular pressure and bleb appearance were evaluated in vivo. The rabbits were sacrificed 8 weeks after the surgery, and their eyes were harvested and processed for tissue analysis. Subsequently, tissue samples were analyzed microscopically for fibrotic tissue and cellular markers of inflammation. Moreover, the collagen-rich fibrotic tissue formed around the stents was analyzed using quantitative histology and infrared spectroscopy. The outcomes of this study were analyzed using the ANOVA test. Results: This study demonstrated no significant differences in intraocular pressure, bleb appearance, or presence of complications such as bleb leak among the treatment groups. In contrast, we observed significant differences among the subpopulations of collagen fibrils formed within scar neo-tissue. Based on the spectroscopic analyses, we determined that the relative content of mature collagen cross-links in the antibody-treated group was significantly reduced compared to other groups. Conclusions: Direct blocking of collagen fibrillogenesis with the anti-collagen antibody offers potentially beneficial effects that may reduce the negative impact of the subconjunctival scarring associated with glaucoma filtering surgery.

Profibrotic behavior of fibroblasts derived from patients that develop posttraumatic shoulder stiffness.

Background and Aims: Arthrofibrosis is a severe scarring condition characterized by joint stiffness and pain. Fundamental to developing arthrofibrotic scars is the accelerated production of procollagen I, a precursor of collagen I molecules that form fibrotic deposits in affected joints. The procollagen I production mechanism comprises numerous elements, including enzymes, protein chaperones, and growth factors. This study aimed to elucidate the differences in the production of vital elements of this mechanism in surgical patients who developed significant posttraumatic arthrofibrosis and those who did not. Methods: We studied a group of patients who underwent shoulder arthroscopic repair of the rotator cuff. Utilizing fibroblasts isolated from the patients' rotator intervals, we analyzed their responses to profibrotic stimulation with transforming growth factor ß1 (TGFß1). We compared TGFß1-dependent changes in the production of procollagen I. We studied auxiliary proteins, prolyl 4-hydroxylase (P4H), and heat shock protein 47 (HSP47), that control procollagen stability and folding. A group of other proteins involved in excessive scar formation, including connective tissue growth factor (CTGF), α smooth muscle actin (αSMA), and fibronectin, was also analyzed. Results: We observed robust TGFß1-dependent increases in the production of CTGF, HSP47, αSMA, procollagen I, and fibronectin in fibroblasts from both groups of patients. In contrast, TGFß1-dependent P4H production increased only in the stiff-shoulder-derived fibroblasts. Conclusion: Results suggest P4H may serve as an element of a mechanism that modulates the fibrotic response after rotator cuff injury.

Circulating inflammatory cytokines alter transcriptional activity within fibrotic tissue of Dupuytren's disease patients.

Dupuytren's disease is a benign fibroproliferative disorder of the hand that results in disabling digital contractures that impair function and diminish the quality of life. The incidence of this disease has been correlated with chronic inflammatory states, but any direct association between inflammatory cytokines and Dupuytren's disease is not known. We hypothesized that advanced fibroproliferation is associated with increased levels of circulating inflammatory cytokines. Blood and fibrotic cord tissue were collected preoperatively from patients with severe contracture and control patients. Blood plasma concentrations of known inflammatory cytokines were evaluated using a multiplex immunoassay. Proteins from the cord tissue were analyzed by RNA sequencing and immunohistochemistry. Moreover, collagen-rich cords were analyzed using Fourier-transform infrared spectroscopy. The results indicate that patients exhibited significantly elevated circulating inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-2, and IL-12p70, as compared with controls. Similarly, IL-4 and IL-13 were detected significantly more frequently in Dupuytren's disease as compared with control. RNA sequencing revealed 5311 differentially expressed genes and distinct clustering between diseased and control samples. In addition to increased expression of genes associated with fibroproliferation, we also observed upregulation of transcripts activated by inflammatory cytokines, including prolactin inducible protein and keratin intermediate filaments. IL-2, but not TNF-α, was detected in fibrotic cord tissue by immunohistochemistry. Finally, spectroscopic assays revealed a significant reduction of the collagen content and alterations of collagen cross-linking within the Dupuytren's disease tissues. In total, our results illustrate that patients with severe Dupuytren's disease exhibit substantially elevated circulating inflammatory cytokines that may drive fibroproliferation. Clinical Significance: The results from this study establish the basis for a specific cytokine profile that may be useful for diagnostic testing and therapeutic intervention in Dupuytren's disease.

Mechanisms of reducing joint stiffness by blocking collagen fibrillogenesis in a rabbit model of posttraumatic arthrofibrosis.

Posttraumatic fibrotic scarring is a significant medical problem that alters the proper functioning of injured tissues. Current methods to reduce posttraumatic fibrosis rely on anti-inflammatory and anti-proliferative agents with broad intracellular targets. As a result, their use is not fully effective and may cause unwanted side effects. Our group previously demonstrated that extracellular collagen fibrillogenesis is a valid and specific target to reduce collagen-rich scar buildup. Our previous studies showed that a rationally designed antibody that binds the C-terminal telopeptide of the α2(I) chain involved in the aggregation of collagen molecules limits fibril assembly in vitro and reduces scar formation in vivo. Here, we have utilized a clinically relevant arthrofibrosis model to study the broad mechanisms of the anti-scarring activity of this antibody. Moreover, we analyzed the effects of targeting collagen fibril formation on the quality of healed joint tissues, including the posterior capsule, patellar tendon, and subchondral bone. Our results show that blocking collagen fibrillogenesis not only reduces collagen content in the scar, but also accelerates the remodeling of healing tissues and changes the collagen fibrils' cross-linking. In total, this study demonstrated that targeting collagen fibrillogenesis to limit arthrofibrosis affects neither the quality of healing of the joint tissues nor disturbs vital tissues and organs.

Collagen-rich deposit formation in the sciatic nerve after injury and surgical repair: A study of collagen-producing cells in a rabbit model.

INTRODUCTION: Posttraumatic scarring of peripheral nerves produces unwanted adhesions that block axonal growth. In the context of surgical nerve repair, the organization of the scar tissue adjacent to conduits used to span the gap between the stumps of transected nerves is poorly understood. The goal of this study was to elucidate the patterns of distribution of collagen-rich scar tissue and analyze the spatial organization of cells that produce fibrotic deposits around and within the conduit's lumen. METHODS: Employing a rabbit model of sciatic nerve transection injury, we studied the formation of collagen-rich scar tissue both inside and outside conduits used to bridge the injury sites. Utilizing quantitative immunohistology and Fourier-transform infrared spectroscopy methods, we measured cellular and structural elements present in the extraneural and the intraneural scar of the proximal and distal nerve fragments. RESULTS: Analysis of cells producing collagen-rich deposits revealed that alpha-smooth muscle actin-positive myofibroblasts were only present in the margins of the stumps. In contrast, heat shock protein 47-positive fibroblasts actively producing collagenous proteins were abundant within the entire scar tissue. The most prominent site of transected sciatic nerves with the highest number of cells actively producing collagen-rich scar was the proximal stump. CONCLUSION: Our findings suggest the proximal region of the injury site plays a prominent role in pro-fibrotic processes associated with the formation of collagen-rich deposits. Moreover, they show that the role of canonical myofibroblasts in peripheral nerve regeneration is limited to wound contracture and that a distinct population of fibroblastic cells produce the collagenous proteins that form scar tissue. As scarring after nerve injury remains a clinical problem with poor outcomes due to incomplete nerve recovery, further elucidation of the cellular and spatial aspects of neural fibrosis will lead to more targeted treatments in the clinical setting.

Increased expression of the transforming growth factor ß-inducible gene HIC-5 in systemic sclerosis skin and fibroblasts: a novel antifibrotic therapeutic target.

OBJECTIVE: SSc is a systemic fibrotic disease affecting skin, numerous internal organs and the microvasculature. The molecular pathogenesis of SSc tissue fibrosis has not been fully elucidated, although TGF-ß1 plays a crucial role. The Hic-5 protein encoded by the TGF-ß1-inducible HIC-5 gene participates in numerous TGF-ß-mediated pathways, however, the role of Hic-5 in SSc fibrosis has not been investigated. The aim of this study was to examine HIC-5 involvement in SSc tissue fibrosis. METHODS: Affected skin from three patients with diffuse SSc and dermal fibroblasts cultured from affected and non-affected SSc skin were examined for HIC-5 and COL1A1 gene expression. Real-time PCR, IF microscopy, western blotting and small interfering RNA-mediated HIC-5 were performed. RESULTS: HIC-5 and COL1A1 transcripts and Hic-5, type 1 collagen (COL1) and α-smooth muscle actin (α-SMA) protein levels were increased in clinically affected SSc skin compared with normal skin and in cultured dermal fibroblasts from affected SSc skin compared with non-affected skin fibroblasts from the same patients. HIC-5 knockdown caused a marked reduction of COL1 production in SSc dermal fibroblasts. CONCLUSION: HIC-5 expression is increased in affected SSc skin compared with skin from normal individuals. Affected SSc skin fibroblasts display increased HIC-5 and COL1A1 expression compared with non-affected skin fibroblasts from the same patients. Hic-5 protein was significantly increased in cultured SSc dermal fibroblasts. HIC-5 mRNA knockdown in SSc fibroblasts caused >50% reduction of COL1 production. Although these are preliminary results owing to the small number of skin samples studied, they indicate that Hic-5 plays a role in the profibrotic activation of SSc dermal fibroblasts and may represent a novel molecular target for antifibrotic therapy in SSc.

The impact of cholesterol deposits on the fibrillar architecture of the Achilles tendon in a rabbit model of hypercholesterolemia.

BACKGROUND: Increased tendon pain and tendon damage is a significant complication related to hyperlipidemia. Unlike the well-established pathogenesis associated with increased serum concentrations of total cholesterol, triglycerides, and low-density lipoprotein in atherosclerotic cardiovascular disease, the role of hyperlipidemia in promoting tendon damage remains controversial and requires mechanistic clarity. METHODS: In this study, we analyzed the consequences of hypercholesterolemia on the integrity of the collagen-based architecture of the Achilles tendon. The Achilles tendons from rabbits fed with normal-cholesterol (nCH) and high-cholesterol (hCH) diets were analyzed. We studied the morphology of tendons, distribution of lipids within their collagen-rich milieu, the relative amounts of fibrillar collagen I and collagen III, and selected biomechanical parameters of the tendons at the macroscale and the nanoscale. RESULTS: Histological assays of hCH tendons and tenosynovium demonstrated hypercellular areas with increased numbers of macrophages infiltrating the tendon structure as compared to the nCH tendons. While Oil Red staining revealed lipid-rich deposits in the hCH tendons, hybridization of tendon tissue with the collagen hybridizing peptide (CHP) demonstrated damage to the collagen fibers. Fourier-transform infrared (FTIR) spectra showed the presence of distinct peaks consistent with the presence of cholesterol ester. Additionally, the hCH tendons displayed regions of poor collagen content that overlapped with lipid-rich regions. The hCH tendons had a substantial fourfold increase in the collage III to collagen I ratio as compared to the nCH tendons. Tendons from the hCH rabbits showed poor biomechanical characteristics in comparison with control. The biomechanical changes were evident at the macrolevel and the nanolevel of tendon structure. CONCLUSIONS: Our findings support the hypothesis that hypercholesterolemia coincides with the weakening of the tendons. It is likely that the intimate contact between collagen fibrils and cholesterol deposits contributes to the weakening of the fibrillar structure of the tendons.

Epiphyseal growth plate architecture is unaffected by early postnatal activation of the expression of R992C collagen II mutant.

Spondyloepiphyseal dysplasia (SED) exemplifies a group of heritable diseases caused by mutations in collagenous proteins of the skeletal system. Its main feature is altered skeletal growth. Pathomechanisms of SED include: changes in the stability of collagen II molecules, inability to form proper collagen fibrils, excessive intracellular retention of mutant molecules, and endoplasmic reticulum stress. The complexity of this pathomechanism presents a challenge for designing therapies for SED. Our earlier research tested whether such therapies only succeed when applied during a limited window of development. Here, employing an inducible mouse model of SED caused by the R992C mutation in collagen II, we corroborate our earlier observations that a therapy must be applied at the prenatal or early postnatal stages of skeletal growth in order to be successful. Moreover, we demonstrate that blocking the expression of the R992C collagen II mutant at the early prenatal stages leads to long-term positive effects. Although, we could not precisely mark the start of the expression of the mutant, these effects are not significantly changed by switching on the mutant production at the early postnatal stages. By demonstrating the need for early therapeutic interventions, our study provides, for the first time, empirically-based directions for designing effective therapies for SED and, quite likely, for other skeletal dysplasias caused by mutations in key macromolecules of the skeletal system.

Engineering the Second Generation of Therapeutic Cells with Enhanced Targeting of Injured Tissues.

Experimental approaches to improving tissue repair utilize cells and growth factors needed to restore the architecture and function of damaged tissues and organs. Key limitations of these approaches include poor delivery of therapeutic cells and growth factors into injury sites, as well as their short-term retention in target areas. In our earlier studies, we demonstrated that artificial collagen-specific anchor (ACSA) expressed on the surface of therapeutic cells directs them into collagen-rich sites of injury. Moreover, we demonstrated that the ACSA improves the retention of these cells in target sites, thereby promoting tissue repair. To advance the ACSA-based technology, we engineered the second generation of the ACSA-expressing cells able to deliver growth factors to target sites. In this study, we specifically focused on insulin growth factor 1 (IGF1), which enhances the repair of a number of collagen-rich connective tissues, including ligament and tendon. Utilizing gene engineering, we produced IGF1 in the ACSA-expressing cells. Using relevant experimental models, we demonstrated that recombinant IGF1 secreted by these cells maintains its specificity and biological activity. Moreover, our studies show that IGF1 produced by the ACSA-expressing cells cultured in three-dimensional environment promotes the formation of the collagen-rich fibrillar matrix. Furthermore, the engineered cells integrated well with the native collagen-rich tendon tissue. Our study provides strong evidence for the great potential of cells with rationally engineered target-specific receptors to restore damaged connective tissues. Future studies in relevant animal models will determine the utility of these cells in vivo.

Target-Specific Delivery of an Antibody That Blocks the Formation of Collagen Deposits in Skin and Lung.

Regardless of the cause of organ fibrosis, its main unwanted consequence is the formation of collagen fibril-rich deposits that hamper the structure and function of affected tissues. Although many strategies have been proposed for the treatment of fibrotic diseases, no therapy has been developed, which can effectively block the formation of collagen fibril deposits. With this in mind, we recently developed an antibody-based therapy to block key interactions that drive collagen molecules into fibrils. In this study, we analyzed target specificity, which is a main parameter that defines the safe use of all antibody-based therapies in humans. We hypothesized that, regardless of the route of administration, our antibody would preferentially bind to free collagen molecules synthesized at the sites of fibrosis and have minimal off-target interactions when applied in various tissues. To test this hypothesis, we used two experimental models of organ fibrosis: (1) a keloid model, in which antibody constructs were directly implanted under the skin of nude mice and (2) an experimental model of pulmonary fibrosis, in which our antibody was administered systemically by intravenous injection. Following administration, we studied the distribution of our antibody within target and off-target sites as well as analyzed its effects on fibrotic tissue formation. We found that local and systemic application of our antibody had high specificity for targeting collagen fibrillogenesis and also appeared safe and therapeutically effective. In summary, this study provides the basis for further testing our antifibrotic antibody in a broad range of disease conditions and suggests that this treatment approach will be effective if delivered by local or systemic administration.

Patterns of production of collagen-rich deposits in peripheral nerves in response to injury: A pilot study in a rabbit model.

INTRODUCTION: Although collagen-rich deposits are the main component of neural scars, the patterns of their formation are ill defined. Essential to the biosynthesis of collagen fibrils are enzymes catalyzing posttranslational modifications and chaperones that control the formation of the collagen triple helix. Prolyl-4-hydroxylase (P4H) and heat shock protein-47 (HSP47) play a key role, and their production is upregulated during scar formation in human tissues. Alpha smooth muscle actin (αSMA) is also produced during fibrotic processes in myofibroblasts that participate in fibrotic response. In injured peripheral nerves, however, the distribution of cells that produce these markers is poorly understood. METHODS: The goal of this study was to determine the distribution of the αSMA-positive, HSP47-positive, and the P4H-positive cells to better understand the formation of collagen-rich fibrotic tissue (FT) in response to peripheral nerve injury. To reach this goal, we employed a rabbit model of crush-injury and partial-transection injury of the sciatic nerves. RESULTS: Our study demonstrated that αSMA is expressed in a relatively small number of cells seen in neural FT. In contrast, cells producing P4H and HSP47 are ubiquitously present in sites of injury of the sciatic nerves. CONCLUSION: We contemplate that these proteins may serve as valuable markers that define fibrotic activities in the injured peripheral nerves.

Prospects and limitations of improving skeletal growth in a mouse model of spondyloepiphyseal dysplasia caused by R992C (p.R1192C) substitution in collagen II.

Skeletal dysplasias form a group of skeletal disorders caused by mutations in macromolecules of cartilage and bone. The severity of skeletal dysplasias ranges from precocious arthropathy to perinatal lethality. Although the pathomechanisms of these disorders are generally well defined, the feasibility of repairing established aberrant skeletal tissues that developed in the presence of mutant molecules is currently unknown. Here, we employed a validated mouse model of spondyloepiphyseal dysplasia (SED) that enables temporal control of the production of the R992C (p.R1192C) collagen II mutant that causes this disease. Although in our earlier studies we determined that blocking the expression of this mutant at the early prenatal stages prevents a SED phenotype, the utility of blocking the R992C collagen II at the postnatal stages is not known. Here, by switching off the expression of R992C collagen II at various postnatal stages of skeletal development, we determined that significant improvements of cartilage and bone morphology were achieved only when blocking the production of the mutant molecules was initiated in newborn mice. Our study indicates that future therapies of skeletal dysplasias may require defining a specific time window when interventions should be applied to be successful.

Blocking collagen fibril formation in injured knees reduces flexion contracture in a rabbit model.

Post-traumatic joint contracture is a frequent orthopaedic complication that limits the movement of injured joints, thereby severely impairing affected patients. Non-surgical and surgical treatments for joint contracture often fail to improve the range of motion. In this study, we tested a hypothesis that limiting the formation of collagen-rich tissue in the capsules of injured joints would reduce the consequences of the fibrotic response and improve joint mobility. We targeted the formation of collagen fibrils, the main component of fibrotic deposits formed within the tissues of injured joints, by employing a relevant rabbit model to test the utility of a custom-engineered antibody. The antibody was delivered directly to the cavities of injured knees in order to block the formation of collagen fibrils produced in response to injury. In comparison to the non-treated control, mechanical tests of the antibody-treated knees demonstrated a significant reduction of flexion contracture. Detailed microscopic and biochemical studies verified that this reduction resulted from the antibody-mediated blocking of the assembly of collagen fibrils. These findings indicate that extracellular processes associated with excessive formation of fibrotic tissue represent a valid target for limiting post-traumatic joint stiffness. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1038-1046, 2017.

Auxiliary proteins that facilitate formation of collagen-rich deposits in the posterior knee capsule in a rabbit-based joint contracture model.

Post-traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen-rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α-subunit of prolyl 4-hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril-stabilizing cross-links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti-fibrotic targets.

Matrix-specific anchors: a new concept for targeted delivery and retention of therapeutic cells.

Biomedical strategies for tissue engineering and repair utilize specific cells, scaffolds, and growth factors to reconstruct elements of damaged tissue. The cellular element of these strategies is limited, however, by poor efficiency of delivery and retention of therapeutic cells in target sites. We propose that the presence of a cellular anchor that is able to specifically bind a defined element of target tissue will facilitate efficient binding and retention of therapeutic cells, thereby promoting repair of the target site. To do so, we engineered an artificial collagen-specific anchor (ACSA) that is able to specifically bind collagen I. The ACSA was engineered by creating a construct comprising rationally designed consecutive domains. The binding specificity of the ACSA was achieved by employing variable regions of a monoclonal antibody that recognizes a unique epitope present in human collagen I. Meanwhile, cell membrane localization of the ACSA was provided by the presence of a transmembrane domain. We determined that the ACSA was localized within cell membranes and interacted with its intended target, that is, collagen I. We have demonstrated that, in comparison to the control, the cells expressing the ACSA attached better to collagen I and exhibited improved retention in sites of seeding. We have also demonstrated that the presence of the ACSA did not interfere with cell proliferation, the biosynthesis of endogenous collagen I, or the biological functions of native collagen receptors. Since the presented cell delivery system utilizes a common characteristic of major connective tissues, namely the presence of collagen I, the findings described here could have a broad positive impact for improving the repair processes of tendon, ligament, bone, intervertebral disc, skin, and other collagen I-rich connective tissues. If successful, the ACSA approach to deliver cells will serve as an outline for developing cell delivery methods that target other elements of extracellular matrices, including other collagen types, laminins, and fibronectins.

Mechanisms of aberrant organization of growth plates in conditional transgenic mouse model of spondyloepiphyseal dysplasia associated with the R992C substitution in collagen II.

Mutations in collagen II, a main structural protein of cartilage, are associated with various forms of spondyloepiphyseal dysplasia (SED), whose main features include aberrations of linear growth. Here, we analyzed the pathomechanisms responsible for growth alterations in transgenic mice with conditional expression of the R992C collagen II mutation. Specifically, we studied the alterations of the growth plates of mutant mice in which chondrocytes lacked their typical columnar arrangement. Our studies demonstrated that chondrocytes expressing the thermolabile R992C mutant collagen II molecules endured endoplasmic reticulum stress, had atypical polarization, and had reduced proliferation. Moreover, we demonstrated aberrant organization and morphology of primary cilia. Analyses of the extracellular collagenous deposits in mice expressing the R992C mutant collagen II molecules indicated their poor formation and distribution. By contrast, transgenic mice expressing wild-type collagen II and mice in which the expression of the transgene encoding the R992C collagen II was switched off were characterized by normal growth, and the morphology of their growth plates was correct. Our study with the use of a conditional mouse SED model not only indicates a direct relation between the observed aberration of skeletal tissues and the presence of mutant collagen II, but also identifies cellular and matrix elements of the pathomechanism of SED.

Testing the anti-fibrotic potential of the single-chain Fv antibody against the α2 C-terminal telopeptide of collagen I.

Abstract This study focuses on the single-chain fragment variable (scFv) variant of the original IgA-type antibody, recognizing the α2 C-terminal telopeptide (α2Ct) of human collagen I, designed to inhibit post-traumatic localized fibrosis via blocking the formation of collagen-rich deposits. We have demonstrated that the scFv construct expressed in yeast cells was able to fold into an immunoglobulin-like conformation, but it was prone to forming soluble aggregates. Functional assays, however, indicate that the scFv construct specifically binds to the α2Ct epitope and inhibits collagen fibril formation both in vitro and in a cell culture model representing tissues that undergo post-traumatic fibrosis. Thus, the presented study demonstrates the potential of the scFv variant to serve as an inhibitor of the excessive formation of collagen-rich fibrotic deposits, and it reveals certain limitations associated with the current stage of development of this antibody construct.