Collagen integrity of the annulus fibrosus in degenerative disc disease individuals quantified with collagen hybridizing peptide.

Introduction: Degenerative disc disease (DDD) is accompanied by structural changes in the intervertebral discs (IVD). Extra-cellular matrix degradation of the annulus fibrosus (AF) has been linked with degeneration of the IVD. Collagen is a vital component of the IVD. Collagen hybridizing peptide (CHP) is an engineered protein that binds to degraded collagen, which we used to quantify collagen damage in AF. This method was used to compare AF samples obtained from donors with no DDD to AF samples from patients undergoing surgery for symptomatic DDD. Methods: Fresh AF tissue was embedded in an optimal cutting temperature compound and cryosectioned at a thickness of 8 µm. Hematoxylin and Eosin staining was performed on sections for general histomorphological assessment. Serial sections were stained with Cy3-conjugated CHP and the mean fluorescence intensity and areal fraction of Cy3-positive staining were averaged for three regions of interest (ROI) on each CHP-stained section. Results: Increases in mean fluorescence intensity (p = 0.0004) and percentage of positively stained area (p = 0.00008) with CHP were detected in DDD samples compared to the non-DDD samples. Significant correlations were observed between mean fluorescence intensity and percentage of positively stained area for both non-DDD (R = 0.98, p = 5E-8) and DDD (R = 0.79, p = 0.0012) samples. No significant differences were detected between sex and the lumbar disc level subgroups of the non-DDD and DDD groups. Only tissue pathology (non-DDD versus DDD) influenced the measured parameters. No three-way interactions between tissue pathology, sex, and lumbar disc level were observed. Discussion and Conclusions: These findings suggest that AF collagen degradation is greater in DDD samples compared to non-DDD samples, as evidenced by the increased CHP staining. Strong positive correlations between the two measured parameters suggest that when collagen degradation occurs, it is detected by this technique and is widespread throughout the tissue. This study provides new insights into the structural alterations associated with collagen degradation in the AF that occur during DDD.

Effect of Different Crosslinkers on Denatured Dentin Collagen's Biostability, MMP Inhibition and Mechanical Properties.

OBJECTIVE: Sound, natural dentin collagen can be stabilized against enzymatic degradation through exogenous crosslinking treatment for durable bonding; however, the effect on denatured dentin (DD) collagen is unknown. Hence, the ability of different crosslinkers to enhance/restore the properties of DD collagen was assessed. METHODS: Demineralized natural and DD collagen films (7 mm × 7 mm × 7 µm) and beams (0.8 mm × 0.8 mm × 7 mm) were prepared. DD collagen was experimentally produced by heat or acid exposure, which was then assessed by various techniques. All specimens were then treated with 1 wt% of chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide (EDC/NHS) and two structurally different flavonoids-theaflavins (TF) from black tea and type-A proanthocyanidins from cranberry juice (CR) for either 30 s or 1 h. The controls were untreated. Dentin films were assessed for chemical interaction and cross-linking effect by FTIR, biostability against exogenous collagenase by weight loss (WL) and hydroxyproline release (HYP), and endogenous matrix metalloproteinases (MMPs) activity by confocal laser microscopy. Dentin beams were evaluated for tensile properties. Data were analyzed using ANOVA and Tukey's test (α = 0.05). RESULTS: Compared with natural collagen, DD collagen showed pronounced structural changes, altered biostability and decreased mechanical properties, which were then improved to various degrees that were dependent on the crosslinkers used, with EDC/NHS being the least effective. Surprisingly, the well-known MMP inhibitor EDC/NHS showed negligible effect on or even increased MMP activity in DD collagen. As compared with control, cross-linking induced by TF and CR significantly increased collagen biostability (reduced WL and HYP release, p < 0.05), MMP inhibition (p < 0.001) and mechanical properties (p < 0.05), regardless of denaturation. CONCLUSIONS: DD collagen cannot or can only minimally be stabilized via EDC/NHS crosslinking; however, the challenging substrate of DD collagen can be enhanced or restored using the promising flavonoids TF and CR.

Targeting collagen damage for sustained in situ antimicrobial activities.

Antimicrobial peptides (AMPs) are promising anti-infective drugs, but their use is restricted by their short-term retention at the infection site, non-targeted uptake, and adverse effects on normal tissues. Since infection often follows an injury (e.g., in a wound bed), directly immobilizing AMPs to the damaged collagenous matrix of the injured tissues may help overcome these limitations by transforming the extracellular matrix microenvironment of the infection site into a natural reservoir of AMPs for sustained in situ release. Here, we developed and demonstrated an AMP-delivery strategy by conjugating a dimeric construct of AMP Feleucin-K3 (Flc) and a collagen hybridizing peptide (CHP), which enabled selective and prolonged anchoring of the Flc-CHP conjugate to the damaged and denatured collagen in the infected wounds in vitro and in vivo. We found that the dimeric Flc and CHP conjugate design preserved the potent and broad-spectrum antimicrobial activities of Flc while significantly enhancing and extending its antimicrobial efficacy in vivo and facilitating tissue repair in a rat wound healing model. Because collagen damage is ubiquitous in almost all injuries and infections, our strategy of targeting collagen damage may open up new avenues for antimicrobial treatments in a range of infected tissues.

Strain-induced collagen denaturation is rate dependent in failure of cerebral arteries.

While soft tissues are commonly damaged by mechanical loading, the manifestation of this damage at the microstructural level is not fully understood. Specifically, while rate-induced stiffening has been previously observed in cerebral arteries, associated changes in microstructural damage patterns following high-rate loading are largely undefined. In this study, we stretched porcine middle cerebral arteries to failure at 0.01 and >150 s-1, both axially and circumferentially, followed by probing for denatured tropocollagen using collagen hybridizing peptide (CHP). We found that collagen fibrils aligned with the loading direction experienced less denaturation following failure tests at high than low rates. Others have demonstrated similar rate dependence in tropocollagen denaturation during soft tissue failure, but this is the first study to quantify this behavior using CHP and to report it for cerebral arteries. These findings may have significant implications for traumatic brain injury and intracranial balloon angioplasty. We additionally observed possible tropocollagen denaturation in vessel layers primarily composed of fibrils transversely aligned to the loading axis. To our knowledge, this is the first observation of collagen denaturation due to transverse loading, but further research is needed to confirm this finding. STATEMENT OF SIGNIFICANCE: Previous work shows that collagen hybridizing peptide (CHP) can be used to identify collagen molecule unfolding and denaturation in mechanically overloaded soft tissues, including the cerebral arteries. But experiments have not explored collagen damage at rates relevant to traumatic brain injury. In this work, we quantified collagen damage in cerebral arteries stretched to failure at both high and low rates. We found that the collagen molecule is less damaged at high than at low rates, suggesting that damage mechanisms of either the collagen molecule or other elements of the collagen superstructure are rate dependent. This work implies that arteries failed at high rates, such as in traumatic brain injury, will have different molecular-level damage patterns than arteries failed at low rates. Consequently, improved understanding of damage characteristics may be expanded in the future to better inform clinically relevant cases of collagen damage such as angioplasty and injury healing.

An Adolescent Murine In Vivo Anterior Cruciate Ligament Overuse Injury Model.

BACKGROUND: Overuse ligament and tendon injuries are prevalent among recreational and competitive adolescent athletes. In vitro studies of the ligament and tendon suggest that mechanical overuse musculoskeletal injuries begin with collagen triple-helix unraveling, leading to collagen laxity and matrix damage. However, there are little in vivo data concerning this mechanism or the physiomechanical response to collagen disruption, particularly regarding the anterior cruciate ligament (ACL). PURPOSE: To develop and validate a novel in vivo animal model for investigating the physiomechanical response to ACL collagen matrix damage accumulation and propagation in the ACL midsubstance, fibrocartilaginous entheses, and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: C57BL/6J adolescent inbred mice underwent 3 moderate to strenuous ACL fatigue loading sessions with a 72-hour recovery between sessions. Before each session, randomly selected subsets of mice (n = 12) were euthanized for quantifying collagen matrix damage (percent collagen unraveling) and ACL mechanics (strength and stiffness). This enabled the quasi-longitudinal assessment of collagen matrix damage accrual and whole tissue mechanical property changes across fatigue sessions. Additionally, all cyclic loading data were quantified to evaluate changes in knee mechanics (stiffness and hysteresis) across fatigue sessions. RESULTS: Moderate to strenuous fatigue loading across 3 sessions led to a 24% weaker (P = .07) and 35% less stiff (P < .01) ACL compared with nonloaded controls. The unraveled collagen densities within the fatigued ACL and entheseal matrices after the second and third sessions were 38% (P < .01) and 15% (P = .02) higher compared with the nonloaded controls. CONCLUSION: This study confirmed the hypothesis that in vivo ACL collagen matrix damage increases with tissue fatigue sessions, adversely impacting ACL mechanical properties. Moreover, the in vivo ACL findings were consistent with in vitro overloading research in humans. CLINICAL RELEVANCE: The outcomes from this study support the use of this model for investigating ACL overuse injuries.

Damaged collagen detected by collagen hybridizing peptide as efficient diagnosis marker for early hepatic fibrosis.

Liver fibrosis is characterized by excessive synthesis and deposition of extracellular matrix (ECM) in liver tissues. However, it still has been lacking of early detection and diagnosis methods. The collagen hybridizing peptide (CHP) is a novel synthetic peptide that enables detection of collagen damage and tissue remodeling. Here, we showed that obvious CHP-positive staining could be detected in the liver while given CCl4 for only 3 days, which was significantly enhanced while given CCl4 for 7 days. However, H&E staining showed no significant changes in fibrous tissue, and sirius red-positive staining could only be observed while given CCl4 for 14 days. Moreover, CHP-positive staining enhanced initially at portal area which further extended into the hepatic lobule, which was increased more significantly than sirius red-positive staining in the model of 10 and 14 days. Further proteomic analysis of CHP-positive staining revealed that pathways associated with ECM remodeling were significantly increased, while retinol metabolism was downregulated. Meanwhile, proteins enriched in cellular gene transcription and signal transduction involved in fibrogenesis were also upregulated, suggesting that fibrosis occurred in CHP-positive staining. Our study provided evidence that CHP could detect the collagen damage in liver, which might be an efficient indicator for the diagnosis of liver fibrosis at a very early stage.

Collagen fibrils from both positional and energy-storing tendons exhibit increased amounts of denatured collagen when stretched beyond the yield point.

Collagen molecules are the base structural unit of tendons, which become denatured during mechanical overload. We recently demonstrated that during tendon stretch, collagen denaturation occurs at the yield point of the stress-strain curve in both positional and energy-storing tendons. We were interested in investigating how this load is transferred throughout the collagen hierarchy, and sought to determine the onset of collagen denaturation when collagen fibrils are stretched. Fibrils are one level above the collagen molecule in the collagen hierarchy, allowing more direct probing of the effect of strain on collagen molecules. We isolated collagen fibrils from both positional and energy-storing tendon types and stretched them using a microelectromechanical system device to various levels of strain. We stained the fibrils with fluorescently labeled collagen hybridizing peptides that specifically bind to denatured collagen, and examined whether samples stretched beyond the yield point of the stress-strain curve exhibited increased amounts of denatured collagen. We found that collagen denaturation in collagen fibrils from both tendon types occurs at the yield point. Greater amounts of denatured collagen were found in post-yield positional fibrils than in energy-storing fibrils. This is despite a greater yield strain and yield stress in fibrils from energy-storing tendons compared to positional tendons. Interestingly, the peak modulus of collagen fibrils from both tendon types was the same. These results are likely explained by the greater crosslink density found in energy-storing tendons compared to positional tendons. The insights gained from this study could help management of tendon and other musculoskeletal injuries by targeting collagen molecular damage at the fibril level. STATEMENT OF SIGNIFICANCE: When tendons are stretched or torn, this can lead to collagen denaturation (damage). Depending on their biomechanical function, tendons are considered positional or energy-storing with different crosslink profiles. By stretching collagen fibrils instead of fascicles from both tendon types, we can more directly examine the effect of tensile stretch on the collagen molecule in tendons. We found that regardless of tendon type, collagen denaturation in fibrils occurs when they are stretched beyond the yield point of the stress-strain curve. This provides insight into how load affects different tendon sub-structures during tendon injuries and failure, which will help clinicians and researchers understand mechanisms of injuries and potentially target collagen molecular damage as a treatment strategy, leading to improved clinical outcomes following injury.

Intra-articular MMP-1 in the spinal facet joint induces sustained pain and neuronal dysregulation in the DRG and spinal cord, and alters ligament kinematics under tensile loading.

Chronic joint pain is a major healthcare challenge with a staggering socioeconomic burden. Pain from synovial joints is mediated by the innervated collagenous capsular ligament that surrounds the joint and encodes nociceptive signals. The interstitial collagenase MMP-1 is elevated in painful joint pathologies and has many roles in collagen regulation and signal transduction. Yet, the role of MMP-1 in mediating nociception in painful joints remains poorly understood. The goal of this study was to determine whether exogenous intra-articular MMP-1 induces pain in the spinal facet joint and to investigate effects of MMP-1 on mediating the capsular ligament's collagen network, biomechanical response, and neuronal regulation. Intra-articular MMP-1 was administered into the cervical C6/C7 facet joints of rats. Mechanical hyperalgesia quantified behavioral sensitivity before, and for 28 days after, injection. On day 28, joint tissue structure was assessed using histology. Multiscale ligament kinematics were defined under tensile loading along with microstructural changes in the collagen network. The amount of degraded collagen in ligaments was quantified and substance P expression assayed in neural tissue since it is a regulatory of nociceptive signaling. Intra-articular MMP-1 induces behavioral sensitivity that is sustained for 28 days (p < 0.01), absent any significant effects on the structure of joint tissues. Yet, there are changes in the ligament's biomechanical and microstructural behavior under load. Ligaments from joints injected with MMP-1 exhibit greater displacement at yield (p = 0.04) and a step-like increase in the number of anomalous reorganization events of the collagen fibers during loading (p ≤ 0.02). Collagen hybridizing peptide, a metric of damaged collagen, is positively correlated with the spread of collagen fibers in the unloaded state after MMP-1 (p = 0.01) and that correlation is maintained throughout the sub-failure regime (p ≤ 0.03). MMP-1 injection increases substance P expression in dorsal root ganglia (p < 0.01) and spinal cord (p < 0.01) neurons. These findings suggest that MMP-1 is a likely mediator of neuronal signaling in joint pain and that MMP-1 presence in the joint space may predispose the capsular ligament to altered responses to loading. MMP-1-mediated pathways may be relevant targets for treating degenerative joint pain in cases with subtle or no evidence of structural degeneration.

Quadruped Gait and Regulation of Apoptotic Factors in Tibiofemoral Joints following Intra-Articular rhPRG4 Injection in Prg4 Null Mice.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4-PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4-/- (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4.

Dentin collagen denaturation status assessed by collagen hybridizing peptide and its effect on bio-stabilization of proanthocyanidins.

OBJECTIVE: To assess dentin collagen denaturation from phosphoric acid and enzyme treatments using collagen hybridizing peptide (CHP) and to investigate the effect of collagen denaturation on bio-stabilization promoted by proanthocyanidins (PA). METHODS: Human molars were sectioned into 7-µm-thick dentin films, demineralized, and assigned to six groups: control with/without PA modification, H3PO4-treated collagen with/without PA modification, enzyme-treated collagen with/without PA modification. PA modification involved immersing collagen films in 0.65% PA for 30 s. H3PO4 and enzyme treatments were used to experimentally induce collagen denaturation, which was quantitated by fluorescence intensity (FI) from the fluorescently-conjugated-CHP (F-CHP) staining (n = 4). FTIR was used to characterize collagen structures. All groups were subject to collagenase digestion to test the bio-stabilization effect of PA on denatured collagen using weight loss analysis and hydroxyproline assay (n = 6). Data were analyzed using two-factor ANOVA and Games-Howell post hoc tests (α = 0.05). RESULTS: FTIR showed collagen secondary structural changes after denaturation treatments and confirmed the incorporation and cross-linking of PA in control and treated collagen. F-CHP staining indicated high-degree, medium-degree, and low-degree collagen denaturation from H3PO4-treatment (FI = 83.22), enzyme-treatment (FI = 36.54), and control (FI = 6.01) respectively. PA modification significantly reduced the weight loss and hydroxyproline release of all groups after digestion (p < 0.0001), with the results correlated with FI values at r = 0.96-0.98. SIGNIFICANCE: A molecular method CHP is introduced as a sensitive technique to quantitate dentin collagen denaturation for the first time. PA modification is shown to effectively stabilize denatured collagen against collagenase digestion, with the stabilization effect negatively associated with the collagen denaturation degree.

Focusing on intramuscular connective tissue: Effect of cooking time and temperature on physical, textual, and structural properties of yak meat.

This study aimed to evaluate the effects of different cooking time (2, 4, and 6 h) and temperature (50, 60, 70, 80, and 90 °C) on physical, textual, and structural properties of longissimus lumborum muscle of yak, and to explore the thermal denaturation process of intramuscular collagen by using a new tool (collagen hybridizing peptide staining, CHP staining). The results showed that tenderness was affected by the interaction of cooking time and temperature and the changes in moisture and collagen composition. In comparison with cooking time, temperature had more obvious effects on cooking loss, moisture content and redness. Scanning electron microscopy showed that as the temperature increased, intramuscular connective tissue gradually degraded, and muscle fibers became more compact. CHP staining showed that the collagen in the perimysium first denatured at 50 °C, and more and more collagen denatured and degraded as the temperature increased.

Adipose Stromal Cell-Secretome Counteracts Profibrotic Signals From IPF Lung Matrices.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by excess deposition and altered structure of extracellular matrix (ECM) in the lungs. The fibrotic ECM is paramount in directing resident cells toward a profibrotic phenotype. Collagens, an important part of the fibrotic ECM, have been shown to be structurally different in IPF. To further understand the disease to develop better treatments, the signals from the ECM that drive fibrosis need to be identified. Adipose tissue-derived stromal cell conditioned medium (ASC-CM) has demonstrated antifibrotic effects in animal studies but has not been tested in human samples yet. In this study, the collagen structural integrity in (fibrotic) lung tissue, its interactions with fibroblasts and effects of ASC-CM treatment hereon were studied. Methods: Native and decellularized lung tissue from patients with IPF and controls were stained for denatured collagen using a collagen hybridizing peptide. Primary lung fibroblasts were seeded into decellularized matrices from IPF and control subjects and cultured for 7 days in the presence or absence of ASC-CM. Reseeded matrices were fixed, stained and analyzed for total tissue deposition and specific protein expression. Results: In both native and decellularized lung tissue, more denatured collagen was observed in IPF tissue compared to control tissue. Upon recellularization with fibroblasts, the presence of denatured collagen was equalized in IPF and control matrices, whereas total ECM was higher in IPF matrices than in the control. Treatment with ASC-CM resulted in less ECM deposition, but did not alter the levels of denatured collagen. Discussion: Our data showed that ASC-CM can inhibit fibrotic ECM-induced profibrotic behavior of fibroblasts. This process was independent of collagen structural integrity. Our findings open up new avenues for ASC-CM to be explored as treatment for IPF.

Mechanical characterisation of the human dura mater, falx cerebri and superior sagittal sinus.

The cranial meninges have been shown to play a pivotal role in traumatic brain injury mechanopathology. However, while the mechanical response of the brain and its many subregions have been studied extensively, the meninges have conventionally been overlooked. This paper presents the first comparative mechanical analysis of human dura mater, falx cerebri and superior sagittal sinus tissues. Biaxial tensile analysis identified that these tissues are mechanically heterogeneous, in contrast to the assumption that the tissues are mechanically homogeneous which is typically employed in FE model design. A thickness of 0.91 ± 0.05 (standard error) mm for the falx cerebri was also identified. This data can aid in improving the biofidelity of the influential falx structure in FE models. Additionally, the use of a collagen hybridizing peptide on the superior sagittal sinus suggests this structure is particularly susceptible to the effects of circumferential stretch, which may have important implications for clinical treatment of dural venous sinus pathologies. Collectively, this research progresses understanding of meningeal mechanical and structural characteristics and may aid in elucidating the behaviour of these tissues in healthy and diseased conditions. STATEMENT OF SIGNIFICANCE: This study presents the first evaluation of human falx cerebri and superior sagittal sinus mechanical, geometrical and structural properties, along with a comparison to cranial dura mater. To mechanically characterise the tissues, biaxial tensile testing is conducted on the tissues. This analysis identifies, for the first time, mechanical stiffness differences between these tissues. Additionally, geometrical analysis identifies that there are thickness differences between the tissues. The evaluation of human meningeal tissues allows for direct implementation of the novel data to finite element head injury models to enable improved biofidelity of these influential structures in traumatic brain injury simulations. This work also identifies that the superior sagittal sinus may be easily damaged during clinical angioplasty procedures, which may inform the treatment of dural sinus pathologies.

Post-translational activation of Mmp2 correlates with patterns of active collagen degradation during the development of the zebrafish tail.

Matrix metalloproteinase-2 (a.k.a. Gelatinase A, or Mmp2 in zebrafish) is known to have roles in pathologies such as arthritis, in which its function is protective, as well as in cancer metastasis, in which it is activated as part of the migration and invasion of metastatic cells. It is also required during development and the regeneration of tissue architecture after wound healing, but its roles in tissue remodelling are not well understood. Gelatinase A is activated post-translationally by proteolytic cleavage, making information about its transcription and even patterns of protein accumulation difficult to relate to biologically relevant activity. Using a transgenic reporter of endogenous Mmp2 activation in zebrafish, we describe its accumulation and post-translational proteolytic activation during the embryonic development of the tail. Though Mmp2 is expressed relatively ubiquitously, it seems to be active only at specific locations and times. Mmp2 is activated robustly in the neural tube and in maturing myotome boundaries. It is also activated in the notochord during body axis straightening, in patches scattered throughout the epidermal epithelium, in the gut, and on cellular protrusions extending from mesenchymal cells in the fin folds. The activation of Mmp2 in the notochord, somite boundaries and fin folds associates with collagen remodelling in the notochord sheath, myotome boundary ECM and actinotrichia respectively. Mmp2 is likely an important effector of ECM remodelling during the morphogenesis of the notochord, a driving structure in vertebrate development. It also appears to function in remodelling the ECM associated with growing epithelia and the maturation of actinotrichia in the fin folds, mediated by mesenchymal cell podosomes.

Collagen denaturation in the infarcted myocardium involves temporally distinct effects of MT1-MMP-dependent proteolysis and mechanical tension.

Tissue injury results in profound alterations in the collagen network, associated with unfolding of the collagen triple helix, proteolytic degradation and generation of fragments. In the infarcted myocardium, changes in the collagen network are critically involved in the pathogenesis of left ventricular rupture, adverse remodeling and chronic dysfunction. We hypothesized that myocardial infarction is associated with temporally and spatially restricted patterns of collagen denaturation that may reflect distinct molecular mechanisms of collagen unfolding. We used a mouse model of non-reperfused myocardial infarction, and in vitro assays in fibroblast-populated collagen lattices. In healing infarcts, labeling with collagen hybridizing peptide (CHP) revealed two distinct patterns of collagen denaturation. During the inflammatory and proliferative phases of infarct healing, collagen denaturation was pericellular, localized in close proximity to macrophages and myofibroblasts. qPCR array analysis of genes associated with matrix remodeling showed that Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) is markedly upregulated in infarct macrophages and fibroblasts, suggesting its involvement in pericellular collagen denaturation. In vitro, MT1-MMP-mediated pericellular collagen denaturation is involved in cardiac fibroblast migration. The effects of MT1-MMP on collagen denaturation and fibroblast migration involve the catalytic site, and require hemopexin domain-mediated actions. In contrast, during the maturation phase of infarct healing, extensive collagen denaturation was noted in the hypocellular infarct, in the infarct border zone and in the mitral valve annulus, in the absence of MT1-MMP. In vitro, mechanical tension in attached collagen lattices was sufficient to induce peripheral collagen denaturation. Our study suggests that in healing infarcts, early pericellular collagen denaturation may be important for migration of macrophages and reparative myofibroblasts in the infarct. Extensive denaturation of collagen fibers is noted in mature scars, likely reflecting mechanical tension. Chronic collagen denaturation may increase susceptibility of the matrix to proteolysis, thus contributing to progressive cardiac dilation and post-infarction heart failure.

Collagen denaturation is initiated upon tissue yield in both positional and energy-storing tendons.

Tendons are collagenous soft tissues that transmit loads between muscles and bones. Depending on their anatomical function, tendons are classified as positional or energy-storing with differing biomechanical and biochemical properties. We recently demonstrated that during monotonic stretch of positional tendons, permanent denatured collagen begins accumulating upon departing the linear region of the stress-strain curve. However, it is unknown if this observation is true during mechanical overload of other types of tendons. Therefore, the purpose of this study was to investigate the onset of collagen denaturation relative to applied strain, and whether it differs between the two tendon types. Rat tail tendon (RTT) fascicles and rat flexor digitorum longus (FDL) tendons represented positional and energy-storing tendons, respectively. The samples were stretched to incremental levels of strain, then stained with fluorescently labeled collagen hybridizing peptides (CHPs); the CHP fluorescence was measured to quantify denatured collagen. Denatured collagen in both positional and energy-storing tendons began to increase at the yield strain, upon leaving the linear region of the stress-strain curve as the sample started to permanently deform. Despite significant differences between the two tendon types, it appears that collagen denaturation is initiated at tissue yield during monotonic stretch, and the fundamental mechanism of failure is the same for the two types of tendons. At tissue failure, positional tendons had double the percentage of denatured collagen compared to energy-storing tendons, with no difference between 0% control groups. These results help to elucidate the etiology of subfailure injury and rupture in functionally distinct tendons.

Advanced glycation end products cause RAGE-dependent annulus fibrosus collagen disruption and loss identified using in situ second harmonic generation imaging in mice intervertebral disk in vivo and in organ culture models.

Aging and diabetes are associated with increased low-back pain and intervertebral disk (IVD) degeneration yet causal mechanisms remain uncertain. Advanced glycation end products (AGEs), which accumulate in IVDs from aging and are implicated in diabetes-related disorders, alter collagen and induce proinflammatory conditions. A need exists for methods that assess IVD collagen quality and degradation in order to better characterize specific structural changes in IVDs due to AGE accumulation and to identify roles for the receptor for AGEs (RAGE). We used multiphoton microscopy with second harmonic generation (SHG), collagen-hybridizing peptide (CHP), and image analysis methods to characterize effects of AGEs and RAGE on collagen quality and quantity in IVD annulus fibrosus (AF). First, we used SHG imaging on thin sections with an in vivo dietary mouse model and determined that high-AGE (H-AGE) diets increased AF fibril disruption and collagen degradation resulting in decreased total collagen content, suggesting an early degenerative cascade. Next, we used in situ SHG imaging with an ex vivo IVD organ culture model of AGE challenge on wild type and RAGE-knockout (RAGE-KO) mice and determined that early degenerative changes to collagen quality and degradation were RAGE dependent. We conclude that AGE accumulation leads to RAGE-dependent collagen disruption in the AF and can initiate molecular and tissue level collagen disruption. Furthermore, SHG and CHP analyzes were sensitive to collagenous alterations at multiple hierarchical levels due to AGE and may be useful in identifying additional contributors to collagen damage in IVD degeneration processes.

Molecular Detection and Assessment of Intervertebral Disc Degeneration via a Collagen Hybridizing Peptide.

During aging, wear, and tear of intervertebral discs, human discs undergo a series of morphological and biochemical changes. Degradation of extracellular matrix proteins, e.g., collagen, arises as an important contributor and accelerator in this process. Existing methods to detect collagen degradation at the tissue level include histology and immunohistochemistry. Unfortunately, most of these methods only depict overall collagen content without the ability to specifically discern degraded collagen and to assess the severity of degeneration. To fill this technological gap, we developed a robust and simple approach to detect and assess early disc degeneration with a collagen hybridizing peptide (CHP) that hybridizes with the flawed triple helix structure in degraded collagen. Intriguingly, the CHP signal in mouse lumbar discs exhibited a linear incremental pattern with age. This finding was corroborated with histological analysis based on established methods. When comparing this analysis, a positive linear correlation was found between CHP fluorescence intensity and the histological score with a regression value of r 2 = 0.9478. In degenerative mouse discs elicited by pro-inflammatory stimuli (IL-1ß and LPS) ex vivo, the newly developed approach empowered prediction of the severity of disc degeneration. We further demonstrated higher CHP signals in a degenerative human disc tissue when compared to a normal sample. These findings also resonated with histological analysis. This approach lays a solid foundation for specific detection and assessment of intervertebral disc degeneration at the molecular level and will promote development of future disc regeneration strategies.

Developing Regenerative Treatments for Developmental Defects, Injuries, and Diseases Using Extracellular Matrix Collagen-Targeting Peptides.

Collagen is the most widespread extracellular matrix (ECM) protein in the body and is important in maintaining the functionality of organs and tissues. Studies have explored interventions using collagen-targeting tissue engineered techniques, using collagen hybridizing or collagen binding peptides, to target or treat dysregulated or injured collagen in developmental defects, injuries, and diseases. Researchers have used collagen-targeting peptides to deliver growth factors, drugs, and genetic materials, to develop bioactive surfaces, and to detect the distribution and status of collagen. All of these approaches have been used for various regenerative medicine applications, including neovascularization, wound healing, and tissue regeneration. In this review, we describe in depth the collagen-targeting approaches for regenerative therapeutics and compare the benefits of using the different molecules for various present and future applications.

Microplate assay for denatured collagen using collagen hybridizing peptides.

The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains (<7.5%) exhibited minimal denatured collagen, but values rapidly increased at medium strains (7.5-10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F-CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay. Further, we achieved these results more efficiently than current methods in a rapid, high-throughput manner, with multiple types of collagenous tissue while maintaining accuracy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:431-438, 2019.