Tendon Collagen Research Articles

Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Preparation and comparison of two medical dressings made from the collagens from fish and bovine.

Collagen is used in medical dressings because of its high hydrophilicity, low immunogenicity, excellent biocompatibility, and degradability. These features can promote cell proliferation and platelet agglomeration. Herein, we studied the preparation of gel dressing by using silver carp skin collagen and bovine collagen as raw materials. Their properties and the application effects of collagen gel dressing were evaluated and compared. The centrifugal stability, rheology, and water-loss rate of silver carp skin collagen gel (SCG) and bovine tendon collagen gel (CTG) were determined. Results showed that the two gels were stable, and SCG had better rheology and ductility than CTG. However, the denaturation temperature and water-retention rate of SCG were slightly lower than those of CTG. Two collagen gels were used in the burn-repair experiment of KM mice. Results showed that the SCG and CTG were consistent with the wound-repair effect of commercially available products for shallow II-degree scald and deep II-degree scald. In the superficial shallow II scald experiment, SCG had a faster healing rate in the first 8 days and a shorter recovery time than CTG. In the deep II-degree scald experiment, the wound-healing rate of SCG on the 14th day reached 94.24%, which was 2 days faster than the recovery time of CTG. Moreover, the skin after wound healing was shallower than the scar produced after CTG treatment. Therefore, SCG had the potential to be used as the medical dressing.

Travelling through the Natural Hierarchies of Type I Collagen with X-rays: From Tendons of Cattle, Horses, Sheep and Pigs.

Type I collagen physiological scaffold for tissue regeneration is considered one of the widely used biomaterials for tissue engineering and medical applications. It is hierarchically organized: five laterally staggered molecules are packed within fibrils, arranged into fascicles and bundles. The structural organization is correlated to the direction and intensity of the forces which can be loaded onto the tissue. For a tissue-specific regeneration, the required macro- and microstructure of a suitable biomaterial has been largely investigated. Conversely, the function of multiscale structural integrity has been much less explored but is crucial for scaffold design and application. In this work, collagen was extracted from different animal sources with protocols that alter its structure. Collagen of tendon shreds excised from cattle, horse, sheep and pig was structurally investigated by wide- and small-angle X-ray scattering techniques, at both molecular and supramolecular scales, and thermo-mechanically with thermal and load-bearing tests. Tendons were selected because of their resistance to chemical degradation and mechanical stresses. The multiscale structural integrity of tendons' collagen was studied in relation to the animal source, anatomic location and source for collagen extraction.

Reduction of Tendon Fibrosis Using Galectin-3 Inhibitors.

Fibrosis is a complication of both tendon injuries and repairs. The authors aimed to develop a mouse model to assess tendon fibrosis and to identify an antifibrotic agent capable of overcoming it. The Achilles tendon of adult C57Bl/6 mice was exposed via skin incision, followed by 50% tendon injury and abrasion with sandpaper. Sham operations were conducted on contralateral hindlimbs. Histologic analyses and immunofluorescent staining for fibrotic markers (collagen type 1 [ Col1 ], α-smooth muscle actin [ α-SMA ]) were used to confirm that the model induced tendon fibrosis. A second experiment further examined the role of α-SMA in adhesion formation using α-SMA.mTmG mice (6 to 8 weeks old; n = 3) with the same injury model. Lastly, α-SMA.mTmG mice were randomized to either condition 1 (tendon injury [control group]) or condition 2 (tendon injury with galectin-3 inhibitor [Gal3i] treatment at time of injury [treatment group]). Histologic analyses confirmed tendon thickening and collagen deposition after tendon injury and abrasion compared with control. Immunofluorescence showed higher levels of Col1 and α-SMA protein expression after injury compared with sham ( P < 0.05). Real-time quantitative polymerase chain reaction also demonstrated increased gene expression of Col1 and α-SMA after injury compared with sham ( P < 0.05). Gal3 protein expression also increased after injury and colocalized with α-SMA+ fibroblasts surrounding the fibrotic tendon. Gal3i treatment decreased collagen deposition and scarring observed in the treatment group ( P < 0.05). The authors' study provides a reproducible and reliable model to investigate tendon fibrosis. Findings suggest the potential of Gal3i to overcome fibrosis resulting from tendon injuries. Tendon injuries are common presentations to hand surgeons. Complications include adhesion formation, which results in reduced strength and frequent reinjury. Advancements in management require a better understanding of the mechanisms behind tendon fibrosis in order to identify ways to overcome it.

Achilles Tendon Tissue Turnover Before and Immediately After an Acute Rupture

Background: An Achilles tendon rupture (ATR) is a frequent injury and results in the activation of tendon cells and collagen expression, but it is unknown to what extent turnover of the tendon matrix is altered before or after a rupture. Purpose/Hypothesis: The purpose of this study was to characterize tendon tissue turnover before and immediately after an acute rupture in patients. It was hypothesized that a rupture would result in pronounced collagen synthesis in the early phase (first 2 weeks) after the injury. Study Design: Cross-sectional study; Level of evidence, 3. Methods: The study included patients (N = 18) eligible for surgery after an ATR. At the time of inclusion, the patients ingested deuterium oxide (2H2O) orally, and on the day of surgery (within 14 days of the injury), they received a 3-hour flood-primed infusion of an 15N-proline tracer. During surgery, the patients had 1 biopsy specimen taken from the ruptured part of the Achilles tendon and 1 that was 3 to 5 cm proximal to the rupture as a control. The biopsy specimens were analyzed for carbon-14 (14C) levels in the tissue to calculate long-term turnover (years), incorporation of 2H-alanine (from 2H2O) into the tissue to calculate the fractional synthesis rate (FSR) of proteins in the short term (days), and incorporation of 15N-proline into the tissue to calculate the acute FSR (hours). Results: Both the rupture and the control samples showed consistently lower levels of 14C compared with the predicted level of 14C in a healthy tendon, which indicated increased tendon turnover in a fraction (48% newly synthesized) of the Achilles tendon already for a prolonged period before the rupture. Over the first days after the rupture, the synthesis rate for collagen was relatively constant, and the average synthesis rate on the day of surgery (2-14 days after the rupture) was 0.025% per hour, irrespective of the length of time after a rupture and the site of sampling (rupture vs control). No differences were found in the FSR between the rupture and control samples in the days after the rupture. Conclusion: Higher than normal tissue turnover in the Achilles tendon before a rupture indicated that changes in the tendon tissue preceded the injury. In addition, we observed no increase in tendon collagen tissue turnover in the first 2 weeks after an ATR. This favors the view that an increase in the formation of new tendon collagen is not an immediate phenomenon during the regeneration of ruptured tendons in patients. Registration: NCT03931486 (ClinicalTrials.gov identifier).

Increased Ca2+ signaling through CaV 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

PIEZO1 gain-of-function gene variant is associated with elevated tendon stiffness in humans.

Prolonged periods of increased physical demands can elicit anabolic tendon adaptations that increase stiffness and mechanical resilience or conversely can lead to pathological processes that deteriorate tendon structural quality with ensuing pain and potential rupture. Although the mechanisms by which tendon mechanical loads regulate tissue adaptation are largely unknown, the ion channel PIEZO1 has been implicated in tendon mechanotransduction, with human carriers of the PIEZO1 gain-of-function variant E756del displaying improved dynamic vertical jump performance compared to noncarriers. Here, we sought to examine whether increased tendon stiffness in humans could explain this increased performance. We assessed tendon morphological and mechanical properties with ultrasound-based techniques in 77 participants of Middle- and West-African descent, and we measured their vertical jumping performance to assess potential functional consequences in the context of high tendon strain-rate loading. Carrying the E756del gene variant (n = 30) was associated with 46.3 ± 68.3% (p = 0.002) and 45.6 ± 69.2% (p < 0.001) higher patellar tendon stiffness and Young's modulus compared to non-carrying controls, respectively. While these tissue level measures strongly corroborate the initial postulate that PIEZO1 plays an integral part in regulating tendon material properties and stiffness in humans, we found no detectable correlation between tendon stiffness and jumping performance in the tested population that comprised individuals of highly diverse physical fitness level, dexterity, and jumping ability.

Biological Safety Assessments of High-Purified Ovine Collagen Type I Biomatrix for Future Therapeutic Product: International Organisation for Standardisation (ISO) and Good Laboratory Practice (GLP) Settings.

Wound care management is incredibly challenging for chronic injuries, despite the availability of various types of wound care products in the market. However, most current wound-healing products do not attempt to mimic the extracellular matrix (ECM) and simply provide a barrier function or wound covering. Collagen is a natural polymer that involves a major constituent of the ECM protein, thus making it attractive to be used in skin tissue regeneration during wound healing. This study aimed to validate the biological safety assessments of ovine tendon collagen type-I (OTC-I) in the accredited laboratory under ISO and GLP settings. It is important to ensure that the biomatrix will not stimulate the immune system to produce any adverse reaction. Therefore, we successfully extracted collagen type-I from the ovine tendon (OTC- I) using a method of low-concentration acetic acid. The three-dimensional (3D) skin patch of spongy OTC-I was a soft and white colour, being tested for safety and biocompatibility evaluations based on ISO 10993-5, ISO 10993-10, ISO 10993-11, ISO 10993-23, USP 40 <151>, and OECD 471. For the dermal sensitisation and acute irritation test, none of the tested animals displayed any erythema or oedema effects (p > 0.005). In addition, there were no abnormalities detected in the organ of the mice after being exposed to OTC-I; additionally, no morbidity and mortality were observed in the acute systemic test under the guideline of ISO 10993-11:2017. The grade 0 (non-reactive) based on ISO 10993-5:2009 was graded for the OTC-I at 100% concentration and the mean number of the revertant colonies did not exceed 2-fold of the 0.9% w/v sodium chloride compared to the tester strains of S. typhimurium (TA100, TA1535, TA98, TA1537), and E. coli (WP2 trp uvrA). Our study revealed that OTC-I biomatrix does not present any adverse effects or abnormalities in the present study's condition of induced skin sensitization effect, mutagenic and cytotoxic towards cells and animals. This biocompatibility assessment demonstrated a good agreement between in vitro and in vivo results regarding the absence of skin irritation and sensitization potential. Therefore, OTC-I biomatrix is a potential medical device candidate for future clinical trials focusing on wound care management.

Spatiotemporal and microstructural characterization of heterotopic ossification in healing rat Achilles tendons.

Achilles tendon rupture is a common debilitating medical condition. The healing process is slow and can be affected by heterotopic ossification (HO), which occurs when pathologic bone-like tissue is deposited instead of the soft collagenous tendon tissue. Little is known about the temporal and spatial progression of HO during Achilles tendon healing. In this study we characterize HO deposition, microstructure, and location at different stages of healing in a rat model. We use phase contrast-enhanced synchrotron microtomography, a state-of-the-art technique that allows 3D imaging at high-resolution of soft biological tissues without invasive or time-consuming sample preparation. The results increase our understanding of HO deposition, from the early inflammatory phase of tendon healing, by showing that the deposition is initiated as early as one week after injury in the distal stump and mostly growing on preinjury HO deposits. Later, more deposits form first in the stumps and then all over the tendon callus, merging into large, calcified structures, which occupy up to 10% of the tendon volume. The HOs were characterized by a looser connective trabecular-like structure and a proteoglycan-rich matrix containing chondrocyte-like cells with lacunae. The study shows the potential of 3D imaging at high-resolution by phase-contrast tomography to better understand ossification in healing tendons.

Thermal analysis of tendon, cartilage and bone samples from shoulder joint replacement patients using differential scanning calorimetry (DSC) and thermogravimetry (DTA/TG)

AbstractDespite advanced imaging techniques, more information on the extent of joint degeneration and modifications in tendon collagen content is required for selecting the appropriate type of shoulder prosthesis. The goal of this study was to compare the thermal parameters of various tissue samples from patients who had shoulder arthroplasty and to correlate the thermal shifts with the outcomes of radiological and histopathological assessments. Rotator cuff (ROC) tendon, hyaline cartilage, and subchondral bone samples were obtained throughout surgery for a comminuted 4-part proximal humerus fracture or cuff tear arthropathy. Thermal parameters were measured using differential scanning calorimetry and thermogravimetry (DTA/TG). ROC: The effect of advanced collagen damage was visible in denaturation curves of osteoarthritis samples. Only moderately increased thermal enthalpy and melting temperature were found in the tissue of the trauma patients. Cartilage: Thermal parameters revealed that structural change generated by pathologic conditions exceeded the effects of trauma. Bone: Thermal parameters from degenerative samples revealed more structural damage and calcification decrease than thermal parameters from trauma samples. Correlation: A statistically significant relationship between the extent of osteoarthritis and the thermal variables of cartilage and subchondral bone was discovered. Authors suggest that thermal analysis may be useful in the diagnostic process of orthopedic and posttraumatic shoulder diseases.

Differences in collagen types in the semitendinosus, quadriceps, and patellar tendons: A report using samples from an 11-year-old patient

BackgroundThis study aimed to determine the differences in the proportions of types I and type III collagen in the semitendinosus tendon (ST), quadriceps tendon (QT), and patellar tendon (PT), which are frequently used as autografts for anterior cruciate ligament (ACL) reconstruction. MethodsOrthopedic surgeons diagnosed habitual dislocation of the left patella and surgically treated an 11-year-old boy. Medial patellofemoral ligament reconstruction, medial patellar tibial ligament reconstruction, and arthroscopic lateral release were performed simultaneously. Tissue samples obtained during treatment that were no longer necessary were used as samples for this study. The samples were fixed, paraffin-embedded, and immunostained for type I and type III collagen. Stained samples were observed under a confocal microscope and evaluated visually and quantitatively to determine the percentages of type I and type III collagen. ResultsVisually, the ST had a higher percentage of type III collagen than the PT and QT. The QT and PT were similar in appearance; both consisted mostly of collagen type I. Quantitative evaluation using images showed that the PT comprised 100% type I collagen. The QT comprised 1% type III collagen. The ST comprised 34% type III collagen. ConclusionIn this patient, the QT and PT had higher percentages of type I collagen, which is considered physically strong. Type III collagen, which is considered physically weak, was most common in the ST. These factors may be associated with the high re-injury rates after ACL reconstruction using the ST for physically immature patients.

Impact of essential amino acid intake, resistance exercise, and aging on the concentration of Achilles peritendinous amino acids and procollagen Iα1 in humans.

Recent studies have shown that consuming amino acid-rich compounds improves tendon collagen content and biomechanical properties. Yet, it is unclear if the consumption of amino acids alters local (peritendinous) amino acid concentrations. If aging or exercise influence local amino acid concentrations in conjunction with an amino acid bolus is also not known. We conducted two studies. In Study 1, young women (n = 7, 25 ± 2 years) completed two identical resistance training sessions with either essential amino acid (EAA) or placebo consumption. In Study 2, an EAA bolus identical to Study 1 was given to younger (n = 7; 27 ± 1year) and older adults (n = 6; 68 ± 2 years). Microdialysis was used to determine Achilles peritendinous amino acid and pro-collagen Iα1 (a marker of collagen synthesis) concentrations. In Study 1, amino acid consumption increased peritendinous concentrations of all EAA except histidine (p < 0.05). In Study 2, the peritendinous concentration of EAAs except for methionine, histidine, and lysine (p > 0.05) increased with time (p < 0.05). Further, the concentrations of most measured amino acids were greater in older adults (p < 0.05). Pro-collagen Iα1 concentration (p > 0.05) was unaffected by exercise, EAA, or aging (p > 0.05). Our findings demonstrate the following: (1) when not combined with exercise, an oral EAA bolus leads to only modest increases in Achilles peritendinous amino acid concentrations; (2) when combined with resistance exercise, EAA consumption resulted in greater peritendinous amino acid concentrations compared to no exercise; (3) the basal concentrations of most amino acids were greater in older adults, and (4) neither the EAA bolus nor exercise altered peritendinous pro-collagen concentrations.

Tendon collagen fibril morphology and nano-mechanics via atomic force microscopy

Due to the hierarchical structure of tendon tissue (fascicles→fibers→fibrils), it is essential to investigate morphology and mechanics at the nano-scopic level to better understand whole body function. Previous studies investigating changes in tendon mechanics after exercise/disease/unloading have focused on the whole tendon and fascicle levels, but limited data exists on adaptations at the collagen fibril level. Objective: Provide a new method to study previously frozen tendon tissue by investigating collagen fibril 1) morphology, 2) nano-mechanical properties, and 3) elasticity maps using atomic force microscopy (AFM). Hypothesis: This method will provide a reliable way to assess tendon structure and function at the nano-scale. Methods: C57BL/6J mouse tendons from NASA Rodent Research-1 (-80°C) were chemically separated and mounted onto glass slides then mechanically separated into tendon subunit sheets and left to air-dry. Collagen fibril morphology was tested with a JPK Nanowizard 4a in tapping mode with a Tap300Al-G cantilever (tip radius=10nm, k=40N/m). Samples were then rehydrated for mechanical testing in Quantitative Imaging (QI) mode with an SNL-10D cantilever (tip radius=2nm, k=0.06N/m). Force data acquisition took place in liquid with a set point of 2nN and Z-speed of 40μm/s. Force-microscopy (FM) and structural data were processed using JPK Data Processing Software and Gwyddion, respectively. Data: Tendon collagen fibrils exhibited uniform D-periods across 38 fibril sections (D-period: 66nm±2.1, diameter: 63nm±23.5). Quantitative analysis of the force curves (n=30) along the highest point of the fibril revealed an average Young’s Modulus (YM) of 2.53MPa±0.31. Elasticity maps (n=3) highlighting the distribution of mechanical properties were generated using FM data. Results Summary: Our tissue preparation protocol combined with AFM reliably determines collagen fibril morphology and nano-mechanical properties of mouse tendons that were previously frozen. As evidence, we present systematic YM within and across different data sets. In addition, the YM values were found to be consistent with previous measurements in tendon tissue under similar conditions. Elasticity maps produced by analysis of FM data indicate the similarity of YM values across the tendon surface. Tendon fibrils were found to have similar D-periodicity despite the variation in fibril size, which is consistent with previous literature. Conclusions: This research provides a new method to test tendon collagen fibril structure and mechanical properties at the nano-scale, presenting a novel analysis (elasticity maps) showing contrast in YM across the fibril surface. Funding: CSUPERB New Investigator Grant to JRB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular Vesicles From Primed Adipose-Derived Stem Cells Enhance Achilles Tendon Repair by Reducing Inflammation and Promoting Intrinsic Healing.

Achilles tendon rupture is a common sports-related injury. Even with advanced clinical treatments, many patients suffer from long-term pain and functional deficits. These unsatisfactory outcomes result primarily from an imbalanced injury response with excessive inflammation and inadequate tendon regeneration. Prior studies showed that extracellular vesicles from inflammation-primed adipose-derived stem cells (iEVs) can attenuate early tendon inflammatory response to injury. It remains to be determined if iEVs can both reduce inflammation and promote regeneration in the later phases of tendon healing and the underlying mechanism. Therefore, this study investigated the mechanistic roles of iEVs in regulating tendon injury response using a mouse Achilles tendon injury and repair model in vivo and iEV-macrophage and iEV-tendon cell coculture models in vitro. Results showed that iEVs promoted tendon anti-inflammatory gene expression and reduced mononuclear cell accumulation to the injury site in the remodeling phase of healing. iEVs also increased collagen deposition in the injury center and promoted tendon structural recovery. Accordingly, mice treated with iEVs showed less peritendinous scar formation, much lower incidence of postoperative tendon gap or rupture, and faster functional recovery compared to untreated mice. Further in vitro studies revealed that iEVs both inhibited macrophage M1 polarization and increased tendon cell proliferation and collagen production. The iEV effects were partially mediated by miR-147-3p, which blocked the toll-like receptor 4/NF-κB signaling pathway that activated the M1 phenotype of macrophages. The combined results demonstrate that iEVs are a promising therapeutic agent that can enhance tendon repair by attenuating inflammation and promoting intrinsic healing.

Functionalised Hybrid Collagen-Elastin for Acellular Cutaneous Substitute Applications.

Wound contracture, which commonly happens after wound healing, may lead to physical distortion, including skin constriction. Therefore, the combination of collagen and elastin as the most abundant extracellular matrix (ECM) skin matrices may provide the best candidate biomaterials for cutaneous wound injury. This study aimed to develop a hybrid scaffold containing green natural resources (ovine tendon collagen type-I and poultry-based elastin) for skin tissue engineering. Briefly, freeze-drying was used to create the hybrid scaffolds, which were then crosslinked with 0.1% (w/v) genipin (GNP). Next, the physical characteristics (pore size, porosity, swelling ratio, biodegradability and mechanical strength) of the microstructure were assessed. Energy dispersive X-ray spectroscopy (EDX) and Fourier transform infrared (FTIR) spectrophotometry were used for the chemical analysis. The findings showed a uniform and interconnected porous structure with acceptable porosity (>60%) and high-water uptake capacity (>1200%), with pore sizes ranging between 127 ± 22 and 245 ± 35 µm. The biodegradation rate of the fabricated scaffold containing 5% elastin was lower (<0.043 mg/h) compared to the control scaffold (collagen only; 0.085 mg/h). Further analysis with EDX identified the main elements of the scaffold: it contained carbon (C) 59.06 ± 1.36-70.66 ± 2.89%, nitrogen (N) 6.02 ± 0.20-7.09 ± 0.69% and oxygen (O) 23.79 ± 0.65-32.93 ± 0.98%. FTIR analysis revealed that collagen and elastin remained in the scaffold and exhibited similar functional amides (amide A: 3316 cm-1, amide B: 2932 cm-1, amide I: 1649 cm-1, amide II: 1549 cm-1 and amide III: 1233 cm-1). The combination of elastin and collagen also produced a positive effect via increased Young's modulus values. No toxic effect was identified, and the hybrid scaffolds significantly supported human skin cell attachment and viability. In conclusion, the fabricated hybrid scaffolds demonstrated optimum physicochemical and mechanical properties and may potentially be used as an acellular skin substitute in wound management.

Focused ultrasound for the remote modulation of nitric oxide release from injectable PEG-fibrinogen hydrogels for tendon repair

Introduction: Tendon disorders such as tendinosis, the degradation of collagen in tendon, or tendonitis, inflammation of tendon tissue, contribute to 30% of musculoskeletal complaints. To address the limitations of currently available treatments for tendon repair, an injectable polyethylene glycol (PEG)-fibrinogen hydrogel encompassing nitric oxide (NO) releasing µ-particles was generated. The release of nitric oxide, a therapeutic molecule that modulates many wound healing processes, from the hydrogel can be modified with thermal and mechanical stimulus. To achieve remote control over NO release from hydrogels after deployment, focused ultrasound (FUS) was explored as it provides highly controlled thermal and mechanical stimulus non-invasively.Methods: In this work, the ability of FUS to remotely elicit on-demand NO generation from acoustically active composite hydrogels via thermal and/or mechanical stimulus was explored. Specifically, the temperature and time-dependent release of NO was simulated and characterized when applying FUS to composite hydrogels.Results: Results from acoustic simulations as well as thermocouple heating studies indicated that high spatial and temporal control over hydrogel warming could be achieved non-invasively with a 3.5 MHz FUS transducer. FUS was also able to remotely control NO release from hydrogels with various thermal magnitudes and durations. Additionally, no apparent changes in the mechanical properties of hydrogels were observed with FUS treatment.Discussion: Utilizing FUS thermal and mechanical stimulus provides a potential method of remotely controlling NO release from hydrogels at a wound site to aid in tendon repair.

Collagen breaks at weak sacrificial bonds taming its mechanoradicals

Collagen is a force-bearing, hierarchical structural protein important to all connective tissue. In tendon collagen, high load even below macroscopic failure level creates mechanoradicals by homolytic bond scission, similar to polymers. The location and type of initial rupture sites critically decide on both the mechanical and chemical impact of these micro-ruptures on the tissue, but are yet to be explored. We here use scale-bridging simulations supported by gel electrophoresis and mass spectrometry to determine breakage points in collagen. We find collagen crosslinks, as opposed to the backbone, to harbor the weakest bonds, with one particular bond in trivalent crosslinks as the most dominant rupture site. We identify this bond as sacrificial, rupturing prior to other bonds while maintaining the material’s integrity. Also, collagen’s weak bonds funnel ruptures such that the potentially harmful mechanoradicals are readily stabilized. Our results suggest this unique failure mode of collagen to be tailored towards combatting an early onset of macroscopic failure and material ageing.

CHARACTERIZATION OF NATIVE AND DECELLULARIZED PORCINE TENDON UNDER TENSION AND COMPRESSION: A CLOSER LOOK AT GLYCOSAMINOGLYCAN CONTRIBUTION TO TENDON MECHANICS

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 &amp; E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p&lt;0.05).Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

Plasma-Polymerised Antibacterial Coating of Ovine Tendon Collagen Type I (OTC) Crosslinked with Genipin (GNP) and Dehydrothermal-Crosslinked (DHT) as a Cutaneous Substitute for Wound Healing

Tissue engineering products have grown in popularity as a therapeutic approach for chronic wounds and burns. However, some drawbacks include additional steps and a lack of antibacterial capacities, both of which need to be addressed to treat wounds effectively. This study aimed to develop an acellular, ready-to-use ovine tendon collagen type I (OTC-I) bioscaffold with an antibacterial coating for the immediate treatment of skin wounds and to prevent infection post-implantation. Two types of crosslinkers, 0.1% genipin (GNP) and dehydrothermal treatment (DHT), were explored to optimise the material strength and biodegradability compared with a non-crosslinked (OTC) control. Carvone plasma polymerisation (ppCar) was conducted to deposit an antibacterial protective coating. Various parameters were performed to investigate the physicochemical properties, mechanical properties, microstructures, biodegradability, thermal stability, surface wettability, antibacterial activity and biocompatibility of the scaffolds on human skin cells between the different crosslinkers, with and without plasma polymerisation. GNP is a better crosslinker than DHT because it demonstrated better physicochemical properties (27.33 ± 5.69% vs. 43 ± 7.64% shrinkage), mechanical properties (0.15 ± 0.15 MPa vs. 0.07 ± 0.08 MPa), swelling (2453 ± 419.2% vs. 1535 ± 392.9%), biodegradation (0.06 ± 0.06 mg/h vs. 0.15 ± 0.16 mg/h), microstructure and biocompatibility. Similarly, its ppCar counterpart, GNPppCar, presents promising results as a biomaterial with enhanced antibacterial properties. Plasma-polymerised carvone on a crosslinked collagen scaffold could also support human skin cell proliferation and viability while preventing infection. Thus, GNPppCar has potential for the rapid treatment of healing wounds.

Connective Tissue Integrity is Lost in Vitamin B-6—Deficient Chicks

The objective of the present investigation was to characterize further the connective tissue disorder produced by pyridoxine (vitamin B-6) deficiency, as previously evidenced by electron microscopy. Following the second post-natal week, fast growing male chicks were deprived of pyridoxine for a 1-mo period. Six weeks post-natally, blood concentrations in the experimental deficiency group had declined to deficiency levels as registered by low concentrations of pyridoxal phosphate (coenzyme form) in erythrocytes, but did not reach levels associated with neurological symptoms. Light microscopic study showed abnormalities in the extracellular matrix of the connective tissues. Collagen cross-links and the aldehyde contents were not significantly lower in cartilage and tendon collagens of vitamin B-6—deficient animals than in age-matched controls; also, their proteoglycan degrading protease and collagenase activities measured in articular cartilages were not greater. Thus, proteolysis was an unlikely alternative mechanism to account for the loss of connective tissue integrity. These results point to the need for further investigation into adhesive properties of collagen associated proteoglycans or other proteins in vitamin B-6—deficient connective tissue.