OBJECTIVE

This review article aims to determine the properties, uses, toxicity, and other side effects of crosslinking agents in tissue scaffolds when applied in vitro and in vivo.

A literature search was performed using the PubMed-NCBI (MEDLINE) database (https://pubmed.ncbi.nlm. nih.gov/) with keywords: crosslinking reagent, collagen, hydroxyapatite, and bone regeneration. GRADE criteria were used to assess the quality of evidence.

A total of six articles were included in the study. Improved mechanical properties of collagen-hydroxyapatite scaffolds with high porosity can be achieved by employing crosslinking methods, including physical dehydrothermal (DHT) treatment, chemical treatment with glutaraldehyde (GA), Microbial Transglutaminase (mTGase), 1‐ethyl‐3‐(3‐ dimethylaminopropyl) carbodiimide (EDAC), or a combination of both DHT and EDAC. Furthermore, the crosslinking of EDAC and DHT can lead to forming ester bonds between activated carboxyl groups and hydroxyl groups.

The combination of DHT and EDAC crosslinking can increase mechanical strength, make the pore size appropriate, make the scaffold more stable, and support cell adhesion so that new cells can grow, and the process of osteogenesis can run more optimally.

OBJECTIVE: To investigate the clinical phenotype and genetic characteristics of a family with Dentinogenesis imperfecta type Ⅰ(DGI-Ⅰ). METHODS: Clinical data were collected from a patient with DGI-Ⅰ admitted to the Reproductive Medicine Department of the Affiliated Hospital of Jining Medical University in March 2024. Clinical and familial data were retrospectively collected. Peripheral blood samples (5 mL each) were obtained from the proband and her family members for genomic DNA extraction, followed by whole-exome sequencing (WES) and Sanger sequencing validation. The pathogenicity of the detected variants was assessed according to the Classification Standards and Guidelines for Genetic Variants formulated by the American Society of Medical Genetics and Genomics (ACMG) (hereinafter referred to as the "ACMG Guidelines"). The study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University (Ethics No. 2024-08-C012), and written informed consent for clinical research were obtained from all participants. RESULTS: The proband, a 35-year-old female, presented with translucent yellow primary teeth and progressive browning, darkening, and loss of permanent teeth, without skeletal abnormalities. Affected family members exhibited similar phenotypes. Genetic testing revealed a heterozygous COL1A2 variant (c.1503+1G>A) in the patient and other affected members, while unaffected family members all lacked this variant. Based on the ACMG Guidelines, this variant was classified as likely pathogenic (PM4 + PP1_Strong + PM2_Supporting). CONCLUSION The COL1A2 c.1503+1G>A heterozygous variant is the disease-causing mutation in this family. Above finding has expanded the mutational spectrum of the COL1A2 gene and provided a basis for genetic counseling and diagnosis in similar cases.
Humans ; Female ; Dentinogenesis Imperfecta/genetics* ; Collagen Type I/genetics* ; Adult ; Pedigree ; Mutation ; Male ; Phenotype ; Exome Sequencing

OBJECTIVE: To explore the genetic etiology of a child with Brain small vessel disease 1 with ocular anomalies. METHODS: A child who was admitted to Ningbo Women and Children's Hospital on May 28, 2022 was selected for the study. Clinical data were collected, and peripheral blood samples from the child and her parents were obtained for genomic DNA extraction. Whole exome sequencing (WES) was performed to screen for pathogenic variants. Candidate variants were validated via Sanger sequencing and subjected to bioinformatic analysis. This study was approved by the Medical Ethics Committee of Ningbo Women and Children's Hospital (Ethics No. EC2020-014). RESULTS: The child was a 7-year-old female with a diagnosis of epilepsy. WES revealed that she has carried a heterozygous missense variant in the COL4A1 gene: c.1792G>A (p.Gly598Ser). Sanger sequencing confirmed that her parents both had the wild-type genotype for this variant. Based on American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines for the Interpretation of Sequence Variants, the variant were predicted to be a likely pathogenic (PS2+PM1+PM2_Supporting+PP3). Bioinformatics predicted that amino acid 598 was highly conserved in different species, formed hydrogen bond with Asp599 after becoming Ser598. CONCLUSION The heterozygous missense variant of the COL4A1 gene c.1792T>C (p.G598S) could be the pathogenic cause of this child with Brain small vessel disease 1 with ocular anomalies.
Humans ; Female ; Child ; Collagen Type IV/genetics* ; Eye Abnormalities/genetics* ; Exome Sequencing ; Mutation, Missense ; Cerebral Small Vessel Diseases/genetics*

OBJECTIVE: To carry out genetic testing and clinical phenotypic characterization on a four-generation Chinese pedigree affected with Stickler syndrome type I and explore its genotype-phenotype correlation. METHODS: A child presented at the Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine in February 2023 for micrognathia, glossoptosis and cleft palate and his family members were selected as the study subjects. Clinical data were collected from the affected members, and peripheral blood samples were obtained from 17 participants (including 4 patients and 13 asymptomatic individuals). Whole exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing. Genotype-phenotype correlation was analyzed by integrating the sequencing data with evidence from existing literature. This study has bee granted by the Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine and Guangzhou Women and Children's Medical Center (Ethics No.: 2022-406B00). RESULTS: The four-generation pedigree has comprised 19 members. In addition to the proband, 5 affected individuals had manifested with high myopia, congenital cataracts, and progressive vision loss. Two deceased members reportedly exhibited similar ocular manifestations. Among the four living patients, two had developed retinal detachment, while two others presented with chronic joint pain onset between 35 ~ 40 years of age. One patient required hip replacement surgery at age 42 secondary to femoral head necrosis. The proband, the youngest affected member, exhibited characteristic phenotypes including congenital micrognathia and cleft palate, consistent with Pierre-Robin syndrome. Genetic analysis revealed a heterozygous nonsense mutation in COL2A1 (NM_001844.5: c.2668C>T; p.Gln890Ter) segregating with the disease in all four symptomatic patients. This variant was absent in asymptomatic family members and unaffected controls. While the mutation is listed in ClinVar, no clinical case report has associated it with this phenotypic spectrum. It was not recorded in population databases (gnomAD v4.1.0, 1000 Genomes Project, or ExAC), supporting its potential pathogenicity. CONCLUSION This study has diagnosed a four-generation Chinese pedigree with Stickler syndrome type I attributed to the pathogenic COL2A1 variant c.2668C>T (p.Gln890Ter), which is a rare nonsense mutation associated with ocular predominance and variable skeletal involvement. Notably, this family exhibited marked clinical heterogeneity despite sharing the identical genotype, which highlighted the challenges in phenotype-genotype correlation. The autosomal dominant transmission pattern observed in this pedigree has provided critical insights into COL2A1-related collagenopathies and underscored the necessity of ultrasonographic monitoring for ocular anomalies during prenatal diagnosis. Above findings have advanced our understanding of the pleiotropic effects in type Ⅱ collagen disorders and laid the foundation for precision-based genetic counseling, enabling targeted cascade screening and implementation of tertiary prevention strategies against congenital disabilities for high-risk families.
Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Arthritis/genetics* ; Collagen Type II/genetics* ; Connective Tissue Diseases/genetics* ; Exome Sequencing ; Genetic Association Studies ; Genotype ; Hearing Loss, Sensorineural/genetics* ; Mutation ; Pedigree ; Phenotype ; Retinal Detachment/genetics* ; East Asian People/genetics*

OBJECTIVE: To explore the clinical phenotype and genetic characteristics in two children with Knobloch syndrome (KNO) due to variants of COL18A1 gene. METHODS: Two children presented at the Genetic Eye Disease Clinic of the Eye Hospital of Wenzhou Medical University in October 2023 for ocular lesions were selected as the study subjects. Relevant clinical data and peripheral venous blood samples were collected from the children and their parents. Following genomic DNA extraction, whole-exome sequencing (WES) was carried out. Candidate variants were verified by Sanger sequencing of the family members. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2021-212-K-185). RESULTS: Both children exhibited characteristic ocular features of KNO including nystagmus, high myopia, and leopard spot fundus. Additionally, child 1 also presented with congenital occipital bone dysplasia and occipital encephalocele, while child 2 was diagnosed with vitreoretinochoroidopathy and bilateral high myopia. WES has identified compound heterozygous variants of the COL18A1 gene in both children, including a c.3013+3A>C splice-site variant and a c.2743C>T (p.Arg915Ter) nonsense variant in child 1, and a novel c.1702-1G>A splice-site variant and a c.3836C>T (p.Ser1279Leu) missense variant in child 2. A comprehensive literature review has identified 63 domestic and international articles involving 167 patients with KNO whom can be classified into three subtypes, with KNO type I being the most common and caused by pathogenic variants in the COL18A1 gene. Both probands in this study were children with KNO type I. Analysis of the genotype-phenotype correlations and population distribution characteristics revealed that the KNO patients exhibited significant clinical and genetic heterogeneity, along with a broad geographic distribution, with a relatively greater number of cases reported in Brazil and China. and a broad geographic distribution, with the highest numbers reported in Brazil and China. While no significant difference in genotype distribution was observed between Chinese and non-Chinese patients, phenotypic disparities were noted, with the non-Chinese cohort showing significantly higher rates of retinal detachment and developmental delay (P < 0.05), whereas Chinese patients exhibited a greater proportion of macular hypoplasia (P < 0.05). CONCLUSION The main clinical manifestations of KNO include high myopia, vitreoretinal dystrophy, and occipital encephalocele. The novel c.1702-1G>A splice-site variant identified in the COL18A1 gene has expanded the mutational spectrum of KNO type I and provided valuable insights for genetic diagnosis, counseling, and clinical management of the disease.
Humans ; Retinal Detachment/congenital* ; Male ; Female ; Child ; Encephalocele/genetics* ; Exome Sequencing ; Collagen Type XVIII/genetics* ; Phenotype ; Retinal Degeneration/genetics* ; Mutation ; Child, Preschool

Five previously undescribed diterpenoids, named succipenoids D‒H (1‒5), along with four undescribed lignans, named succignans A‒D (6‒9), were isolated from the dichloromethane extract of Chinese medicinal succinum. Compounds 1‒5 were characterized as nor-abietane diterpenoids, while compounds 6‒9 were identified as lignans polymerized from two groups of phenylpropanoid units. The structures of these novel compounds, including their absolute configurations, were determined through spectroscopic and computational methods. Biological assessments of renal fibrosis demonstrated that compounds 6 and 7 effectively reduce the expression of proteins associated with renal fibrosis, including α-smooth muscle actin (α-SMA), collagen I, and fibronectin in transforming growth factor-β1 (TGF-β1) induced normal rat kidney proximal tubular epithelial cells (NRK-52e).
Animals ; Rats ; Lignans/isolation & purification* ; Diterpenes/isolation & purification* ; Fibrosis/drug therapy* ; Drugs, Chinese Herbal/pharmacology* ; Molecular Structure ; Cell Line ; Kidney Diseases/pathology* ; Transforming Growth Factor beta1/genetics* ; Kidney/metabolism* ; Actins/genetics* ; Fibronectins/genetics* ; Collagen Type I/genetics* ; Epithelial Cells/metabolism*

Objective To explore the mechanism by which gentiopicroside (GPS) prevents macrophage-mediated hepatic fibrosis by regulating the macrophage migration inhibitory factor (MIF)-secreted phosphoprotein 1 (SPP1) signaling pathway in hepatic stellate cells. Methods LX-2 cells were divided into control group, transforming growth factor β(TGF-β) group, and TGF-β combined with GPS (25, 50, 100, 150 μmol/mL) groups. Cell proliferation was detected by EDU assay, cell invasion was assessed by Transwell^TM assay, and the protein expressions of α-smooth muscle actin (α-SMA) and type I collagen (COL1A1) were measured by Western blot. M1-type macrophage-conditioned medium (M1-CM) was used to treat LX-2 cells in the TGF-β group and TGF-β combined with GPS group. The concentrations of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in the cell supernatant, as well as cell proliferation, invasion ability, and the expressions of α-SMA and COL1A1 were detected. Bioinformatics analysis was performed to identify the target intersections of GPS, hepatic fibrosis, and macrophage-related genes. Drug affinity responsive target stability (DARTS) experiments and Western blot were used to verify the regulatory effect of GPS on MIF. Furthermore, LX-2 cells were divided into control group, TGF-β group, TGF-β combined with M2-CM group, TGF-β and oe-NC combined with M2-CM group, and TGF-β and oe-MIF combined with M2-CM group to analyze the concentrations of iNOS and Arg1 in the cell supernatant, as well as changes in cell proliferation, invasion, and the expressions of α-SMA and COL1A1. LX-2 cells were also divided into control group, TGF-β group, TGF-β combined with oe-NC group, TGF-β combined with oe-MIF group, and TGF-β and oe-MIF combined with GPS group to determine the protein expressions of MIF and SPP1 by Western blot. A rat model of hepatic fibrosis was constructed to explore the potential therapeutic effects of GPS on hepatic fibrosis in vivo. Results Compared with the control group, the proliferation and invasion abilities of LX-2 cells in the TGF-β group were increased, and the protein expressions of α-SMA and COL1A1 were enhanced. GPS intervention inhibited the proliferation and invasion of LX-2 cells under TGF-β conditions and reduced the expressions of α-SMA and COL1A1. Compared with the control group, the concentration of iNOS in the cell supernatant of the TGF-β group was upregulated, while the concentration of Arg1 was decreased. M1-CM treatment further increased the concentration of iNOS, decreased the concentration of Arg1, and promoted cell proliferation and invasion, as well as upregulated the expressions of α-SMA and COL1A1 on the basis of TGF-β intervention. However, GPS could reverse the effects of M1-CM intervention. Bioinformatics analysis revealed that MIF was one of the target intersections of GPS, hepatic fibrosis, and macrophage-related genes, and GPS could target and inhibit its expression. Compared with the TGF-β group, after M2-CM intervention, the concentration of iNOS in the cell supernatant decreased, the concentration of Arg1 increased, the proliferation and invasion abilities of LX-2 cells were reduced, and the expressions of α-SMA and COL1A1 were weakened. However, overexpression of MIF reversed the effects of M2-CM intervention. Western blot results showed that compared with the control group, the protein expressions of MIF and SPP1 were enhanced in the TGF-β group. Overexpression of MIF further enhanced the expressions of MIF and SPP1, while GPS intervention inhibited the expressions of MIF and SPP1. In the animal experiment, GPS intervention treatment alleviated liver injury in rats with hepatic fibrosis and inhibited the expressions of MIF and SPP1, as well as α-SMA and COL1A1 in liver tissue. Conclusion GPS may prevent macrophage-mediated hepatic fibrosis by inhibiting the MIF-SPP1 signaling pathway in hepatic stellate cells.
Hepatic Stellate Cells/metabolism* ; Signal Transduction/drug effects* ; Macrophage Migration-Inhibitory Factors/genetics* ; Liver Cirrhosis/prevention & control* ; Macrophages/drug effects* ; Iridoid Glucosides/pharmacology* ; Humans ; Cell Proliferation/drug effects* ; Animals ; Cell Line ; Collagen Type I/metabolism* ; Collagen Type I, alpha 1 Chain ; Intramolecular Oxidoreductases/genetics* ; Rats ; Transforming Growth Factor beta/pharmacology* ; Actins/metabolism*

OBJECTIVES: To compare the clinical effects of concentrated growth factor (CGF) membrane and Bio-Gide ^® collagen membrane, combined with Bio-Oss ^® sticky bone respectively in alveolar ridge preservation (ARP) of maxillary anterior teeth. METHODS: Thirty patients who needed alveolar ridge preservation after maxillary anterior tooth extraction were selected and randomly assigned to the Bio-Gide group and the CGF group. In both groups, the extraction sockets were tightly filled with the Bio-Oss^® sticky bone. In the Bio-Gide group used Bio-Gide^® collagen membrane to cover the upper edge of the Bio-Oss^® sticky bone and closed the wound. The CGF group, the CGF membrane was covered on the upper edge of the Bio-Oss^® sticky bone and the wound was closed. The soft tissue wound healing status at 10 days after ARP, the changes in alveolar ridge height and width immediately after ARP and at 6 months after ARP, and the doctor-patient satisfaction at 6 months after ARP were compared and evaluated between the two groups. RESULTS: At 6 months after ARP, there was no statistically significant difference in the changes of alveolar bone width and height between the two groups (P>0.05). However, the CGF group showed better performance in soft tissue healing after ARP and doctor-patient satisfaction, and the differences were statistically significant (P<0.05). CONCLUSIONS Compared with the Bio-Gide^® collagen membrane, the combined application of CGF membrane and Bio-Oss^® sticky bone can lead to better soft tissue healing after ARP of maxillary anterior teeth and higher doctor-patient satisfaction, showing obvious advantages in ARP of maxillary anterior teeth.
Humans ; Maxilla/surgery* ; Tooth Extraction ; Alveolar Process/surgery* ; Membranes, Artificial ; Alveolar Ridge Augmentation/methods* ; Intercellular Signaling Peptides and Proteins/therapeutic use* ; Minerals/therapeutic use* ; Collagen ; Wound Healing ; Tooth Socket/surgery* ; Bone Substitutes/therapeutic use* ; Male ; Female ; Middle Aged ; Alveolar Bone Loss/prevention & control* ; Adult

Collagen, a vital matrix protein for various tissue and functions in animals, is widely applied in biomaterials. In type Ⅰ collagen, missense mutations of glycine (Gly) in the Gly-Xaa-Yaa triplet of the triple helix are a major cause of osteogenesis imperfecta (OI). Clinical manifestations exhibit marked heterogeneity, spanning a broad disease spectrum from mild skeletal fragility (Type Ⅰ) to severe limb deformities (Type Ⅲ) and perinatal lethal forms (Type Ⅱ). This study utilized recombinant collagen as a model to further elucidate whether Gly→Ala/Val mutations at the N-terminus of the integrin-binding sequence GFPGER affect collagen structure and function, and to explore the underlying mechanisms by which missense mutations impact the biological function of collagen. By introducing Ala and Val substitutions at seven Gly positions N-terminal to the GFPGER sequence, we systematically assessed the effects of these amino acid replacements on the triple-helical structure, thermal stability, integrin-binding ability, and cell adhesion of recombinant collagen. All constructs formed a stable triple-helix structure, with slightly compromised thermal stability. Gly→Val substitutions increased the susceptibility of recombinant collagen to trypsin, which suggested local conformational perturbations in the triple helix. In addition, Gly→Val substitutions significantly reduced the integrin-binding affinity and decreased HT1080 cell adhesion, with the effects stronger than Gly→Ala substitutions. Compared with Gly→Ala substitutions, substitution of Gly with the larger residue Val had enhanced negative effects on the structure and function of recombinant collagen. These findings provide new insights into the molecular mechanisms of osteogenesis imperfecta and offer theoretical references and experimental foundations for the design of collagen sequences and the development of collagen-based biomaterials.
Recombinant Proteins/biosynthesis* ; Glycine/genetics* ; Humans ; Osteogenesis Imperfecta/genetics* ; Integrins/metabolism* ; Collagen/metabolism* ; Collagen Type I/metabolism* ; Amino Acid Substitution ; Mutation ; Mutation, Missense

Scar formation is characterized by dynamic alterations in collagen secretion, which critically determine scar morphology and pathological progression. In fibroblasts, collagen secretion is initiated through the activation of cytokine- and integrin-mediated signaling pathways, which promote collagen gene transcription. The procollagen polypeptide α chains undergo extensive post-translational modifications, including hydroxylation and glycosylation, within the endoplasmic reticulum (ER), followed by folding and assembly into triple-helical procollagen. Subsequent intracellular trafficking involves the sequential transport of procollagen through the ER, Golgi apparatus, and plasma membrane, accompanied by further structural refinements prior to extracellular secretion. Once secreted, procollagen is enzymatically processed to form mature collagen fibrils, which drive scar tissue remodeling. Recent advances in elucidating regulation of collagen secretion have identified pivotal molecular targets, such as transforming growth factor-beta 1 (TGF-β1), prolyl 4-hydroxylase (P4H), heat shock protein 47 (HSP47), and transport and Golgi organization protein 1 (TANGO1), providing novel therapeutic strategies to mitigate pathological scar hyperplasia and improve regenerative outcomes. This review provides a comprehensive analysis of the molecular mechanisms governing collagen secretion during scar formation, with emphasis on signaling cascades, procollagen biosynthesis, intracellular transport dynamics, and post-translational modifications, thereby offering a framework for developing targeted anti-scar therapies.
Humans ; Collagen/metabolism* ; Cicatrix/pathology* ; Signal Transduction ; Transforming Growth Factor beta1/metabolism* ; Fibroblasts/metabolism* ; Animals