Collagen Research Articles

Mechanism of collagen type IV regulation by focal adhesion kinase during retained fetal membranes in dairy cows

Retained fetal membranes (RFM) is an important reproductive disease in dairy cows, caused by maternal and fetal placental tissue adhesion. The main collagen in maternal and fetal placenta tissues is collagen type IV (COL-IV) and its breakdown is the key to placental expulsion. Focal adhesion kinase (FAK) has been shown to regulate the hydrolysis of Col-IV by affecting the activity of MMP-2 and MMP-9 activity, but the regulation of the mechanisms involved in placenta expulsion in dairy cows after postpartum are still unclear. The aim of this study was to investigate the pathogenic mechanism of RFM by studying the relationship between the FAK signaling pathway and COL-IV regulation. Maternal placental tissues were collected from six healthy and six cows with RFM of similar age, parity, body condition and milk yield at 12 h postpartum. In vitro experiments were performed on bovine endometrial epithelial cells from three groups including a FAK inhibitor group, a FAK activator group and a control group without FAK inhibitor and activator. The abundance of molecules involved in the FAK signaling pathway and COL-IV was detected by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The immunohistochemical results showed that the key molecules of FAK signaling pathway FAK, Src, MMP-2 and MMP-9 and Col-IV were expressed in placental tissues. The expression level of FAK, Src, MMP-2, and MMP-9 were significantly down-regulated (P < 0.05) and the abundances of COL-IV were significantly up-regulated (P < 0.05) in maternal placental tissues of RFM cows compared with healthy cows. In the FAK inhibitor treatment group, the relative expression levels of FAK and other related proteins were significantly down-regulated (P < 0.05) and the relative expression levels of COL-IV were significantly up-regulated (P < 0.05) with the results of the FAK activation group the opposite. These results indicated that FAK in maternal endometrial epithelial cells could regulate the hydrolysis process of Col-IV through the expression of key factors of signaling pathways and promote collagen hydrolysis, which in turn facilitated the process of postpartum placenta expulsion in dairy cows.

Chitosan-glucose derivative membrane obtained by Maillard reaction improves cartilage repair in a rabbit model

BackgroundTreatment of articular cartilage injury remains a challenging clinical problem in orthopedics. Chitosan-derived biomaterial could be a potential adjuvant treatment to improve cartilage repair. In the current study, we examined the effects of two potential chitosan-derived materials on cartilage regeneration of osteochondral defects in rabbits.MethodsAn osteochondral defect was created over the rabbit knee and treated using three approaches: group A received no material (n = 24), group B received chitosan membranes with glucose absorption (CGA; n = 25), and group C received chitosan-glucose derivative membranes obtained via the Maillard reaction (CGMR; n = 25). Cartilage repair over the osteochondral defect was analyzed 12 weeks post-surgery via histological analysis, immunostaining, and reverse transcription-qualitative polymerase chain reaction (RT-qPCR) for type-I and type-II collagen mRNA.ResultsAccording to histological analysis, CGMR-treated defects showed significantly improved modified O’Driscoll scoring when compared with no material- and CGA-treated defects (20.9 ± 4.3 vs. 13.00 ± 2.5 and 17.7 ± 4.6, p < 0.001). Moreover, group C exhibited higher intensity of type-II collagen immunohistochemical staining over the regenerated cartilage than groups A and B, along with increased expression of type-II collagen mRNA by RT-qPCR.ConclusionsCGMR might improve cartilage regeneration in osteochondral defects.

Flexible high electrochemical collagen/lignin composite hydrogel for sensing and supercapacitor applications

Synthesis of polymer-based highly conductive hydrogels from natural and renewable sources with robust mechanical performances in flexible electronics remains a great challenge. In this research, a dynamic redox system is designed by using collagen (CL), sulfonate lignin (SL), acrylic acid (AA), and Al3+ to synthesize CL/PAA/SL/Al hydrogels. The formation of effective complexes of Al3+ with the abundant functional groups of CL, SL and PAA, the prepared hydrogel delivers various specific properties, for example, excellent ionic conductivity (4.61 S·m−1), stretchability and antimicrobial performance. The CL/PAA/SL/Al hydrogel demonstrates good mechanical strength, while the maximum tensile strength of the hydrogels is ∼604 kPa at a stretching of 1254 %, and the maximum compressive strength is ∼0.45 MPa, with the maximum stretching of 59.6 %. The CL/PAA/SL/Al hydrogel acts as a flexible strain sensor with high sensitivity. Enough hydroxyl and carboxyl groups in the hydrogels are essential for delivering the maximum 191 mV of open circuit voltage (Voc) rendered during moisture spraying. The supercapacitor assembled from CL/PAA/SL/Al hydrogel manifests specific capacitance (Cs), maximum energy density (Ed) and power density (Pd) of 268.75 F·g−1, 23.89 Wh·kg−1 and 2.4 kW·kg−1, respectively. The supercapacitor can retain its capacitance of 95.8 % after 5000 consecutive charge-discharge cycles.

Proanthocyanidins modification of the mineralized collagen scaffold based on synchronous self-assembly/mineralization for bone regeneration

Proteoglycans (PG) is crucial for regulating collagen formation and mineralization during bone tissue development. A wide variety of PG-modified collagen scaffolds have been proposed for bone engineering application to promote biological responses and work as artificial matrices that guide tissue regeneration. However, poor performance of theses biomaterials against infections has led to an unmet need for clinical prevention. Therefore, we utilized proanthocyanidins (PA) to simulate the functions of PG, including mediating the collagen assembly and intrafibrillar mineralization, to optimize scaffolds performance. The excellent antibacterial properties of PA can endow the scaffolds with anti-infection effects in the process of tissue regeneration. When PA was added during fibrillogenesis, the collagen fibrils appeared irregular aggregation and the mineralization degree was reduced. In contrast, the addition of PA after collagen self-assembly improved the latter’s ability to act as a deposition template and remarkably promoted mineral ions infiltration, thus enhancing intrafibrillar mineralization. The PA-modified scaffold displayed a highly hydrophilicity behaviour and long-term resistance to degradation. The sustained release of PA effectively inhibited the activity of Staphylococcus aureus. The scaffold also showed excellent biocompatibility and improved bone regeneration in calvarial critical-size defect models. The application of PA enables a dual-function scaffold with favourable intrafibrillar mineralization and anti-bacterial properties for bone regeneration.

A-050 Reference Intervals for Plasma Concentrations of C-Terminal Telopeptide of Type-I Collagen (CTX) for Greek Adult Population

Abstract Background CTX assays measure a fragment of the C-terminal telopeptide of type I collagen released during resorption of mature bone. To adequately interpret CTX results, method-specific and population specific reference intervals are needed. We aimed to determine reference intervals for plasma concentrations of CTX. Methods We collected samples from 431 apparently healthy Greeks (140 men as well as 150 pre- and 139 postmenopausal women), who volunteered to participate in our study. Data on socio-demographic characteristics, medical histories and medications were collected and subjects with conditions or receiving medications that affecting bone metabolism were excluded. Dual-energy x-ray absorptiometry (DXA) used to measure bone mineral density (BMD) in all participants. All blood collections were performed in the morning after overnight fast. Plasma EDTA samples were used for the measurement of CTX. Measurements were performed by an automated chemiluminescent immunoassay on the Maglumi-X3 analyzer (Snibe, Shenzhen, China). The reference interval was defined as the central 95% range and determined according to CLSI guide C28-A3 using the MedCalc Software. Results The table summarizes our results. The mean age of men, pre- and post-menopausal women were 50.7, 40,7 and 58.4 years respectively. DXA results revealed that 348 participants had normal BMD, 74 HAD osteopenia and 9 had osteoporosis which excluded from analysis. Since our data were not normally distributed (Shapiro-Wilk test) in any group and after the exclusion of outliers (Tukey test), we used the non-parametric method suggested by the CLSI guide to determine the reference intervals. Multiple regression analysis also revealed that sex menopausal status and BMD were factors affecting CTX values. Conclusions We provide reference intervals for the determination of CTX concentrations in EDTA-plasma for the Greek adult population using an automated immunoassay. Our data may aid to interpret bone turnover in the Greek adult population.

A-112 Reference Intervals for Serum Concentrations of N-Terminal Propeptide of Type-I Procollagen (PINP) for Greek Adult Population

Abstract Background PINP is a sensitive indicator of the synthesis of type-I collagen. Serum PINP levels are affected by changes in bone metabolism and is a useful indicator of disease activity in Paget's disease, bone metastases of osteoblastic nature and in monitoring osteoporosis treatment. To interpret PINP, method and population specific reference intervals are needed. We aimed to determine reference intervals for serum concentrations of PINP for the Greek adult population. Methods We collected samples from 431 apparently healthy Greeks (140 men as well as 150 pre- and 139 post-menopausal women), who volunteered to participate in our study. Data on socio-demographic characteristics, medical histories and medications were collected and subjects with conditions or receiving medications that affecting bone metabolism were excluded. Dual-energy x-ray absorptiometry (DXA) used to measure bone mineral density (BMD) in all participants. All blood collections were performed in the morning after overnight fast. Measurements were performed by an automated chemiluminescent immunoassay on the Maglumi-X3 analyzer (Snibe, Shenzhen, China). This method measures the total-PINP in serum. The reference interval was defined as the central 95% range and determined according to CLSI guide C28-A3 using the MedCalc Software Results The table summarizes our results. DXA results revealed that 348 participants had normal BMD, 74 had osteopenia and 9 had osteoporosis which excluded from analysis. Since our data were not normally distributed (Shapiro-Wilk test) in any group and after the exclusion of outliers (Tukey test), we used the non-parametric method suggested by the CLSI guide in order to determine the reference intervals. Multiple regression analysis revealed that sex and menopausal status were factors affecting PINP values. Conclusions We provide reference intervals for the determination of PINP concentrations in serum for the Greek adult population using an automated immunoassay. Our data may aid to interpret bone turnover in the Greek adult population.

A-049 Effect of Supplementation With Prebiotics on Levels of 25-Hydroxy Vitamin D and on Biomarkers of Bone Remodelling-Results From the OSTEOME Clinical Trial

Abstract Background It is proven that intestinal microbes regulate bone metabolism and can affect the quality of the bone. Prebiotics contain no microorganisms, only substances stimulating their growth. Moreover, flavonoids and catechines are transformed by the microbes into bioactive compounds and polyphenols can modify the microflora composition. OSTEOME is a randomized double-blinded clinical trial to evaluate the efficacy of dietary supplements enriched with prebiotics, in advancing the role of gut microbiota in bone remodelling. We present the effect of supplementation with prebiotics on the serum levels of 25-hydroxy vitamin D (25OHD) and on levels bone markers Osteocalcin (OCN), and C-Terminal Telopeptide of Type-I Collagen (CTX). Methods We enrolled 120 postmenopausal women with osteopenia. Participants were randomized into three groups of 40-participants. Group-A received a supplement containing 600mg calcium citrate and 2400IUof vitamin D3 only, group-B received a supplement additionally containing flavonoids (120mg Naringin and 120mg Hesperidine) and group-C received additionally prebiotic oligosaccharides (2000mg fructooligosaccharides and acacia gum). Blood collections were performed at baseline and at 3, 6 and 9 months for the measurement of bone remodelling biomarkers. Levels of serum Calcium (sCa), 25OHD, as well as levels bone markers OCN, and CTX were measured at all timepoints of follow-up. Results The mean age of group-A, group-B and group-C participants was 60.72(+8.08), 62.37(+7.70) and 62.09(+7.09) (p=NS), and the mean BMIs were 25.35(+3.96), 25.89(+3.80) and 26.25(+4.56) (p=NS). All women were osteopenic as mean total hip T-score was -1.282(+0.617), -1.193(+0.659) and -1.273(+0.573) at baseline for all three groups respectively. The results of OCN, CTX and 25OHD are shown in the table. Conclusions Only patients in group-C showed significant changes in CTX and 25OHD at 9-months, and OCN at 6- and 9-months. Addition of prebiotics to supplements might improve the vitamin D absorption from the gut and give positive balance to biomarkers of bone metabolism.

Coenzyme Q10 and Vitamin E Regulate the Bioactivity of Human Corneal Fibroblast Cells.

Purpose: Corneal fibroblasts are involved in the wound healing of the cornea with proliferation, migration, and differentiation processes. Coenzyme Q10 (CoQ10) and vitamin E can enhance corneal wound healing when applied after a corneal lesion as an eye drop. Thus, this study was performed to determine the potential efficiency of a CoQ10 ophthalmical solution containing a CoQ10 and vitamin E D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-derived formulation in human corneal fibroblasts (HCFs) in vitro. Methods: Primary HCFs were obtained from cadaveric corneal tissue, and cell viability was determined using MTT assay at 24 and 72 h. Cell migration was evaluated using an in vitro wound healing assay, and mRNA expressions of collagen type I (COL-I), collagen type III (COL-III), lumican, hyaluronan, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitors of MMP (TIMP)-1, TIMP-2, interleukin (IL)-1β, IL-6, IL-8, and IL-10 were assessed using reverse transcription polymerase chain reaction at 24 and 72 h. Results: At various concentrations of CoQ10 ophthalmical solution (CoQ10-os), cell viability and wound healing rates of HCFs increased compared with the control group. The expressions of COL-I, COL-III, lumican, and hyaluronan were increased by CoQ10-os, whereas those of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were not affected by CoQ10-os at 24 and 72 h. In treating HCFs with a CoQ10-os medium, IL-1β, IL-6, and IL-8 decreased, whereas IL-10 was significantly increased in a time- and dose-dependent manner. Conclusions: The findings indicate that CoQ10 and vitamin E-TPGS are potent regulators of the bioactivity of HCFs, thus supporting their potential application as ophthalmical solutions in therapies aimed at the fast regeneration of damaged cornea tissues.

Bone strength and residual compressive stress in apatite crystals

Residual stresses are omnipresent in composite materials due to fabrication processes. Residual compressive stresses were recently also observed to develop in collagen fibrils during mineralization and residual strains have been reported in a range of bony materials spanning tooth dentin to mammalian and fish bones. Treatments by heat or by irradiation show that compressive residual stresses up the 100 MPa can be released in the mineral by inducing damage to the collagen fibers. This mini-review assembles some of the knowledge about residual stresses in bony nanocomposites and uses a composite model to argue that residual stresses play a major role in enhancing the strength of bone.

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

Tinglu Yixin granule inhibited fibroblast-myofibroblast transdifferentiation to ameliorate myocardial fibrosis in diabetic mice

Ethnopharmacological relevanceMyocardial fibrosis is one of the pathological characteristics of advanced diabetic cardiomyopathy (DCM) and serves as the strong evidence of poor prognosis. Among them, the transdifferentiation of cardiac fibroblasts (CFs) may play a crucial role in the development of myocardial fibrosis in DCM. Tinglu Yixin granule (TLYXG) has been clinically used for many years and can significantly improve cardiac function of patients with DCM. However, the effect of TLYXG on myocardial fibrosis in DCM remains unknown, and the underlying mechanisms of its efficacy have yet to be fully understood. Aim of the studyThis study aimed to investigate the impact and underlying mechanism of TLYXG on myocardial fibrosis in diabetes mice. Materials and methodsThe bioactive compounds in TLYXG were identified using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). The potential mechanism of TLYXG in treating DCM was predicted using network pharmacology combined with molecular docking and protein-protein docking. The mice model of type 2 diabetes were established by intraperitoneal injection of streptozotocin (STZ) and the high-fat diet (HFD). Indicators of pancreatic islet function, lipids, oxidative stress, and inflammatory factors were tested using kits. Cardiac function was assessed in diabetic mice using echocardiography. Histologic staining was performed to evaluate myocardial hypertrophy and fibrosis. Mechanistically, the hypothesis was tested through rescue experiments. The expression levels of transient receptor potential channel 6 (TRPC6), transforming growth factor-β1 (TGF-β1), collagen I (COL-I) and alpha-smooth muscle actin (α-SMA), along with the mRNA and phosphorylation levels of SMAD family member 3 (Smad3) and protein 38 mitogen-activated protein kinase (p38 MAPK), were assessed using quantitative RT-qPCR, western blot, immunohistochemistry, and immunofluorescence. Neonatal lactating mice were used to extract primary CFs for vitro experiments. Scratch and transwell assays were conducted to assess CFs migration and invasion abilities. Western blot and immunofluorescence were used to evaluate the expression levels of CFs transdifferentiation markers COL-I and α-SMA. ResultsA total of 168 active ingredients were detected in TLYXG based on UPLC-MS and databases. Network pharmacology indicated that TLYXG could improve DCM through inflammatory mediator regulation of TRP channels, TGF-beta signaling pathway, and MAPK signaling pathway. ELISA results showed that TLYXG could ameliorate metabolic levels, inflammation, and oxidative stress in diabetic mice. Echocardiography suggested that TLYXG improved cardiac systolic and diastolic dysfunction in diabetic mice. Histological analysis revealed that TLYXG alleviated myocardial fibrosis in diabetes mice. Additionally, molecular docking analysis indicated strong binding activity between the main active ingredients of TLYXG and TRPC6 of the TRP family. At the molecular level, TLYXG reduced the mRNA and protein expression levels of TRPC6 and TGF-β1 and inhibited the mRNA and phosphorylation levels of Smad3 and p38 MAPK. Furthermore, TLYXG inhibited CFs migration and invasion, and reduced the expression levels of the CFs transdifferentiation markers COL-I and α-SMA. ConclusionTLYXG inhibited the proliferation, migration, invasion and transdifferentiation of CFs by suppressing TGF-β1/Smad3/p38 MAPK signaling through down-regulation of TRPC6, thereby ameliorating myocardial fibrosis in diabetes mice.

TLR-2 and TLR-4 mRNA expression in different grades of histopathological lesions of equine endometrium from follicular phase.

Increased synthesis and deposition of collagen(COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll-like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen-associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K-D) grading and COL1 expression during the follicular phase of the oestrous cycle. Forty samples of endometrial tissues were collected post-mortem from mares on the follicular phase of the oestrous cycle (10 samples of each K-D category). Relative mRNA transcription of TLR-2, TLR-4 and COL1A2 genes was assessed using qPCR, and COL1 protein expression by Western blot analysis. The COL1A2 transcription increased in category IIB when compared to categories I, IIA and III endometria (p < .01). The relative protein abundance of COL1 showed no significant differences between endometrial categories (p > .05). As for the TLRs mRNA transcription, TLR-2/-4 transcripts increased in IIA when compared to the other K-D endometria categories (p < .05). Our findings suggest that TLRs may be involved in the initiation of the endometrial inflammatory response. Additional studies are needed to explore TLRs' potential role as diagnostic markers for monitoring inflammation progression and fibrosis development, as well as their involvement in the mechanisms underlying fibrotic pathways.

Type V collagen exhibits distinct regulatory activities in TMJ articular disc versus condylar cartilage during postnatal growth and remodeling

Understanding matrix molecular activities that regulate the postnatal growth and remodeling of the temporomandibular joint (TMJ) articular disc and condylar cartilage will enable the development of effective regenerative strategies targeting TMJ disorders. This study elucidated the distinct roles of type V collagen (collagen V) in regulating these two units. Studying the TMJ of young adult Col5a1+/− mice, we found that loss of collagen V resulted in substantial changes in the proliferation, clustering and density of progenitors in condylar cartilage, but did not have a major impact on disc cells that are more fibroblast-like. Although loss of collagen V led to thickened collagen fibrils with increased heterogeneity in the disc, there were no significant changes in local micromodulus, except for a reduction at the posterior end of the inferior side. Following the induction of aberrant occlusal loading by the unilateral anterior crossbite (UAC) procedure, both wild-type (WT) and Col5a1+/− condylar cartilage exhibited salient remodeling, and Col5a1+/− condyle developed more pronounced degeneration and tissue hypertrophy at the posterior end than the WT. In contrast, neither UAC nor collagen V deficiency induced marked changes in the morphology or biomechanical properties of the disc. Together, our findings highlight the distinct roles of collagen V in regulating these two units during postnatal growth and remodeling, emphasizing its more crucial role in condylar cartilage due to its impact on the highly mechanosensitive progenitors. These results provide the foundation for using collagen V to improve the regeneration of TMJ and the care of patients with TMJ disorders. Statement of significanceSuccessful regeneration of the temporomandibular joint (TMJ) articular disc and condylar cartilage remains a significant challenge due to the limited understanding of matrix molecular activities that regulate the formation and remodeling of these tissues. This study demonstrates that collagen V plays distinct and critical roles in these processes. In condylar cartilage, collagen V is essential for regulating progenitor cell fate and maintaining matrix integrity. In the disc, collagen V also regulates fibril structure and local micromechanics, but has a limited impact on cell phenotype or its remodeling response. Our findings establish collagen V as a key component in maintaining the integrity of these two units, with a more crucial role in condylar cartilage due to its impact on progenitor cell activities.

Riboflavin-ultraviolet-A collagen crosslinking treatments in improving dentin bonding and resistance to enzymatic digestion

Background/purposeThe efficacy of riboflavin-ultraviolet-A (RF-UVA) treatment in crosslinking collagen and improving dentin bonding has been proven. However, biodegradation of the hybrid layer may compromise the bonding. The purpose of this study was to evaluate different RF-UVA treatments regarding their ability to preserve dentin bonding from enzymatic digestion. Materials and methodsCollagen subjected to different RF (0.1 %, 1 %)-UVA (1, 2, 5 min) treatments and 5 % glutaraldehyde (GA), without or with enzymatic digestion, were examined by gel electrophoresis (SDS-PAGE). Twenty-five teeth with exposed dentin were primed with one of three RF-UVA treatments (0.1 %RF/1-minUVA, 0.1 %RF/2-minUVA, and 1 %RF/1-minUVA), GA, or distilled water after acid-etching, then restored with an adhesive and a resin composite. After 24-h storage, these teeth were sectioned into microbeams. Half of them received an early microtensile bond strength (μTBS) test, while the other half was stored in enzyme solution for 7 days before testing. Nanoleakage and hybrid layer degradation were examined by TEM. ResultsAccording to SDS-PAGE results, all groups showed the dissipation of intense γ bands of collagen after digestion. For the early bonded specimens and after enzymatic digestions, 0.1 %RF/2-minUVA treated group presented the highest μTBS and none of premature failure. Its TEM images showed less nanoleakage after digestion, which is contributed to the well suspended collagen fibrils and resin infiltration in the hybrid layer. ConclusionRF-UVA treatment attained collagen crosslinking effects to improve resin-dentin bonding. 0.1 %RF/2-minUVA effectively enhanced dentin bond strength and resistance to enzymatic digestion by optimally expanding dentinal collagen matrix to facilitate hybrid layer formation.

Colloidal platinum nanoparticles enhance resin-dentin bonding durability

ObjectivesThis study aims to investigate the effect of colloidal platinum nanoparticles (CPN) on the durability of resin-dentin bonding performance with contemporary adhesives. MethodsSixty non-carious human maxillary premolars were subjected to microtensile bond strength (µTBS) testing and divided into two main groups: CPN-treated and untreated. Within each group, specimens were randomly allocated to Clearfil Megabond 2 (MB2), Scotchbond Universal Plus Adhesive with self-etch mode (SE-SUP), and etch-and-rinse mode (ER-SUP) subgroups (n = 10/group). CPN was applied to dentin in the MB2 and SE-SUP groups for 20 s, followed by rinsing before adhesive application. In the ER-SUP group, CPN was applied after etch-and-rinse. The µTBS was tested after 24 h, 6 months, and 1 year, and the fracture modes were observed using SEM. The µTBS data were analyzed using a two-way ANOVA and post-hoc Tukey HSD test (α = 0.05). An additional twelve premolars underwent TEM/STEM/EDX for ultra-morphological observations. ResultsThe application of CPN significantly prevented a decline in the µTBS of both the MB2 and SE-SUP groups. No significant decrease was observed in the ER-SUP group, either with aging or CPN application. Ultra-morphological images revealed platinum nanoparticles attaching to the collagen fibrils of the hybrid layer regardless of aging. It was highlighted that the nanoparticles attached to the banded collagen in the aging groups were observed. SignificanceCPN exhibits the potential in enhancing the longevity of resin-dentin bonding in SE mode.

Effect of Quadriceps Tendon Autograft Preparation and Fixation on Graft Laxity During Suspensory Anterior Cruciate Ligament Reconstruction: A Biomechanical Analysis.

Favorable collagen fibril density and thickness combined with advances in graft preparation and fixation have significantly increased interest in the quadriceps tendon (QT) autograft for anterior cruciate ligament (ACL) reconstruction. While various suspensory techniques have been described, the biomechanical profile of these constructs is largely undefined. To compare the biomechanics of suspensory techniques for soft tissue QT autograft fixation in an in vitro model of ACL reconstruction. Controlled laboratory study. Full-thickness QT grafts were harvested using a 9-mm graft blade. Adjustable-loop devices (ALDs) were secured to the graft (n = 6 per group) with a combination implant containing the ALD and suture tape-reinforced whipstitching (tape-reinforced [TR] group), tethered superficially to the graft with a whipstitch (onlay [OL] group), luggage-tagged through and around the graft (luggage tag [LT] group), or staggered behind superficial suturing (staggered [SG] group). Grafts were tested on an electromechanical testing machine following a validated in vitro reconstruction model of intraoperative workflow and postoperative ACL kinematics, cyclic loading, and load to failure. The TR group had significantly less postcyclic tension loss (mean, 24%) compared with the OL (56%; P = .002), LT (69%; P < .001), and SG (90%; P < .001) constructs. Cyclic elongation was below the 3.0-mm threshold defined as clinical failure for TR (1.6 mm), but not for OL (3.3 mm), LT (7.9 mm), and SG (11.3 mm). All constructs were within native ACL stiffness limits (220 ± 72 N/mm) without significant differences. Ultimate loads significantly exceeded a normal ACL loading limit of 454 N for TR (739 N; P = .023), OL (547 N; P = .020), and LT (769 N; P = .001), but not for SG (346 N; P = .236). The TR ALD construct demonstrated the most favorable time-zero biomechanical properties of modern soft tissue QT suspensory constructs, with 32% less tension loss and 52% less cyclic elongation versus the closest construct. Failure loading of all constructs was acceptable with respect to the native ACL except for the SG group, which had suboptimal ultimate load. TR ALD implants may protect soft tissue QT autografts before graft-bone healing in ACL reconstruction by minimizing time-zero laxity and fixation failure.

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2−/− mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2−/− (double knockout) mice resembled Phlpp1−/− mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1−/− and Phlpp1/2−/− chondrocytes had similarities and were significantly different from WT and Phlpp2−/− chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.

Scaffolds for Osteochondral Lesions of the Talus: Systematic Review and Meta-Analysis of the Last Ten Years Literature.

Scaffolds are widely used devices for the treatment of osteochondral lesions of the talus (OCLT), aimed at enhancing mechanical stability and fostering chondrogenic differentiation. A systematic review and meta-analysis were performed to evaluate the safety, and clinical and radiological results of scaffolds for OCLT management. On 2 January 2024, a search was performed in four databases (PubMed, Embase, Web of Science, and Scopus), according to PRISMA guidelines. The risk of bias in the included studies was also evaluated. Thirty clinical studies were included in the qualitative analysis: 12 retrospective case series, 3 retrospective comparative studies, 9 prospective case series, 1 prospective comparative study, and 1 Randomized Controlled Trial (RCT). Natural scaffolds, such as bilayer collagen (COLL)I/III and hyaluronic scaffolds, were the most employed. Only minor adverse events were observed, even if more serious complications were shown, especially after medial malleolar osteotomy. An overall clinical and radiological improvement was observed after a mean of 36.3 months of follow-up. Patient age and Body Mass Index (BMI), lesion size, and location were correlated with the clinical outcomes, while meta-analysis revealed significant improvement in clinical scores with hyaluronic scaffolds compared to microfracture alone. This study highlights the safety and positive clinical outcomes associated with the use of scaffolds for OCLT. In the few available comparative studies, scaffolds have also demonstrated superior clinical outcomes compared to microfractures alone. Nevertheless, the analysis has shown the limitations of the current literature, characterized by an overall low quality and scarcity of RCTs.

Study on Printability Evaluation of Alginate/Silk Fibroin/Collagen Double-Cross-Linked Inks and the Properties of 3D Printed Constructs.

In recent years, biological 3D printing has garnered increasing attention for tissue and organ repair. The challenge with 3D-printing inks is to combine mechanical properties as well as biocompatibility. Proteins serve as vital structural components in living systems, and utilizing protein-based inks can ensure that the materials maintain the necessary biological activity. In this study, we incorporated two natural biomaterials, silk fibroin (SF) and collagen (COL), into a low-concentration sodium alginate (SA) solution to create novel composite inks. SF and COL were modified with glycidyl methacrylate (GMA) to impart photo-cross-linking properties. The UV light test and 1H NMR results demonstrated successful curing of silk fibroin (SF) and collagen (COL) after modification and grafting. Subsequently, the printability of modified silk fibroin (RSFMA)/SA with varying concentration gradients was assessed using a set of three consecutive printing models, and the material's properties were tested. The research results prove that the addition of RSFMA and ColMA enhances the printability of low-concentration SA solutions, with the Pr values increasing from 0.85 ± 0.02 to 0.90 ± 0.03 and 0.92 ± 0.02, respectively, and the mechanical strength increasing from 0.19 ± 0.01 to 0.28 ± 0.01 and 0.38 ± 0.01 MPa; cytocompatibility has also been improved. Furthermore, rheological tests indicated that all of the inks exhibited shear thinning properties. CCK-8 experiments demonstrated that the addition of ColMA increased the cytocompatibility of the ink system. Overall, the utilization of SF and COL-modified SA materials as inks represents a promising advancement in 3D-printed ink technology.

Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model.

This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated. (1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining." The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.