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Background: Gene transfer of the transcription factor SOX9 with clinically adapted recombinant adeno-associated virus (rAAV) vectors offers a powerful tool to durably enhance the repair process at sites of osteochondral injuries and counteract the development of perifocal osteoarthritis (OA) in the adjacent articular cartilage. Purpose: To examine the ability of an rAAV sox9 construct to improve the repair of focal osteochondral defects and oppose perifocal OA development over time in a large translational model relative to control gene transfer. Study Design: Controlled laboratory study. Methods: Standardized osteochondral defects created in the knee joints of adult sheep were treated with rAAV-FLAG-h sox9 relative to control (reporter) rAAV- lacZ gene transfer. Osteochondral repair and degenerative changes in the adjacent cartilage were monitored using macroscopic, histological, immunohistological, and biochemical evaluations after 6 months. The microarchitecture of the subchondral bone was assessed by micro–computed tomography. Results: Effective, prolonged sox9 overexpression via rAAV was significantly achieved in the defects after 6 months versus rAAV- lacZ treatment. The application of rAAV-FLAG-h sox9 improved the individual parameters of defect filling, matrix staining, cellular morphology, defect architecture, surface architecture, subchondral bone, and tidemark as well as the overall score of cartilage repair in the defects compared with rAAV- lacZ. The overexpression of sox9 led to higher levels of proteoglycan production, stronger type II collagen deposition, and reduced type I collagen immunoreactivity in the sox9- versus lacZ-treated defects, together with decreased cell densities and DNA content. rAAV-FLAG-h sox9 enhanced semiquantitative histological subchondral bone repair, while the microstructure of the incompletely restored subchondral bone in the sox9 defects was not different from that in the lacZ defects. The articular cartilage adjacent to the sox9-treated defects showed reduced histological signs of perifocal OA changes versus rAAV- lacZ. Conclusion: rAAV-mediated sox9 gene transfer enhanced osteochondral repair in sheep after 6 months and reduced perifocal OA changes. These results underline the potential of rAAV-FLAG-h sox9 as a therapeutic tool to treat cartilage defects and afford protection against OA. Clinical Relevance: The delivery of therapeutic rAAV sox9 to sites of focal injuries may offer a novel, convenient tool to enhance the repair of osteochondral defects involving both the articular cartilage and the underlying subchondral bone and provide a protective role by reducing the extent of perifocal OA.

Background: Cell-free devices have been introduced to restore osteochondral defects, avoiding the limitations of cell-based procedures. Among these, an osteochondral scaffold made of type I collagen and hydroxyapatite has been investigated with promising results up to medium-term follow-up. However, the clinical and imaging results over time still need to be documented. Purpose: To evaluate the clinical outcome and tissue maturation at long-term follow-up after the implantation of the osteochondral scaffold. Study Design: Case series; Level of evidence, 4. Methods: A total of 24 patients (7 women, 17 men; age, 36 ± 9.5 years) underwent surgical implantation of the osteochondral scaffold and were prospectively evaluated before surgery, at 2-, 5-, and 10-year follow-up. The mean defect size was 2.9 ± 1.4 cm2. Patients were evaluated using the International Knee Documentation Committee (IKDC) subjective and objective scores, and the activity level was documented with the Tegner score. Magnetic resonance imaging (MRI) evaluation involved the use of the magnetic resonance observation of cartilage repair tissue score combined with 5 more variables focused on the bone layer. Results: A statistically significant improvement of all clinical scores was documented from the baseline to the final evaluation. The IKDC subjective score improved from the preoperative level to 2 years (41 ± 13.2 and 77.1 ± 14.6, respectively) ( P < .0005), with stable results up to 10 years (77.4 ± 19.4). The IKDC objective score changed from 52% of normal and nearly normal knees before the treatment to 84% at 10 years ( P < .0005). Tegner sports activity at the final evaluation (3.8 ± 1.7) was higher compared with the preoperative level (1.6 ± 1.1; P < .05), but it remained significantly lower compared with the preinjury level (5.5 ± 2.6; P < .05). Treatment failed in 1 patient. Persisting graft alterations were observed on MRI scans, although without correlating with the clinical outcome. Conclusion: The regenerative potential of this scaffold is limited, as demonstrated by the signal alterations persisting over time on MRI scans. On the other hand, the clinical improvement was significant and stable over time both in terms of subjective and objective outcomes, including activity level, with overall good results.

Background Measurement of procollagen type I N-terminal propeptide (PINP) concentration in serum reflects the rate of type I collagen synthesis and can therefore be used as a bone formation marker. There are two methods of PINP quantification; the first measures the trimeric propeptide (intact PINP) and the second measures both the trimeric and monomeric propeptides (total PINP). Trimeric PINP is excreted via hepatic endothelial cells, whereas monomeric PINP is cleared renally. Therefore, in renal failure, the total assay has a positive bias with respect to the intact assay, due to monomeric PINP accumulation. The aim of this study was to compare the performance of both assays across all stages of chronic kidney disease. Methods Serum was taken from male ( n = 111) and female ( n = 105) patients attending a metabolic bone clinic, and these were partitioned into stages of chronic kidney disease 1–5. Each serum sample was analysed using the Roche electrochemiluminescence immunoassay for total PINP and the Immunodiagnostic Systems chemiluminescence immunoassay for intact PINP. Results Passing-Bablok regression analysis comparing both methods showed that with advancing chronic kidney disease there was a proportional positive bias affecting the total assay when compared with the intact assay. This proportional positive bias was statistically significant for chronic kidney disease stages 3b, 4 and 5. Conclusions Based on this method comparison study, usage of the total PINP assay should be avoided in chronic kidney disease stages 3b, 4 and 5 (eGFR ≤44 mL/min/1.73 m2) and instead an intact assay used as the total assay overestimates PINP concentrations due to monomeric PINP accumulation.

Current adhesives bond to dentin via a micro-interlocking mechanism within the hybrid layer. Besides such mechanical retention, bonding to dentin would benefit from additional chemical interaction between collagen and resin. This study aims to synthesize a novel light-curable collagen crosslinker methacrylate (MA) functionalized grapeseed extract (GSE) and to assess MAGSE’s ability to crosslink dentin collagen in a clinically relevant setting as well as its role in light-cure as a resin. MA functionalization was accomplished by reacting GSE with methacryloyl chloride to obtain MAGSE, which was characterized by 1H-NMR and Fourier transformed infrared spectroscopy (FTIR). The 6-µm-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, they were treated for 30 s by 1% MAGSE. Collagen crosslinking and resistance to digestion of MAGSE were evaluated by FTIR, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) assay of films, and scanning electron microscopy (SEM)/transmission electron microscopy (TEM) on slabs. Meanwhile, 1% MAGSE or GSE was added to an experimental adhesive formulated with 2-hydroxyethyl methacrylate and a tricomponent photoinitiator system. Polymerization kinetics were monitored continuously in real time for 10 min using FTIR–attenuated total reflection. The results indicated that MAGSE could bind to dentin collagen and protect it from collagenase degradation as strong as GSE. Dentin collagen treated by 1% MAGSE for 30 s was scarcely digested (1.6 ± 1.6%) after 1 h in 0.1% collagenase, while untreated collagen was completely digested (100.9 ± 20.2%). SEM/TEM images indicated MAGSE efficiently crosslinked dentin collagen in 30 s and rendered it almost inert to digestion under clinically relevant settings. Unlike GSE that hindered light-curing of HEMA, MAGSE accelerated the rate of polymerization and exhibited typical traits of a resin monomer with multiple polymerizable units. In conclusion, a novel collagen crosslinking resin MAGSE is synthesized, which inherits collagen crosslinking ability from GSE and polymerization function from MA. Inclusion of this light-curable collagen crosslinker into adhesives might be a revolutionary way to improve durability of dentin bonding in composite restorations.

Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococci to dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients.

Background Serum total N-terminal propeptide of type I collagen (P1NP) and serum C-terminal telopeptide of type I collagen (CTX) are used as reference analytes of bone turnover in clinical application. Biological variation is important for clinical application of these biomarkers. However, the biological variation data of these biomarkers are not consistent. The present study determined biological variations of total P1NP and CTX and their confidence intervals in China using electrochemiluminescence. Methods We collected samples from 25 healthy individuals (17 women and 8 men, ranging from 22 to 49 years of age) at weekly intervals for six weeks. Samples were analysed in a single run in duplicate. Biological variations and their related parameters, such as reference change value and index of individuality (II) were calculated. The results were compared with individual studies in the EFLM database. Results Within-subject and between-subject biological variations were 8.0% and 32.5% for total P1NP and 11.4% and 38.7% for CTX, respectively. The index of individuality for total P1NP and CTX was 0.25 and 0.30, while the reference change value for P1NP values and CTX was 22.4% and 31.9%, respectively. Conclusions No difference was found in weekly biological variation of bone turnover markers between men and premenopausal women. Compared with daily and monthly variation, the present study based on weekly variation provided additional support for clinical application.

Idiopathic epiretinal membranes are sheets of tissue that develop in the vitreoretinal interface. They are formed by cells and extracellular matrix, and they are considered the expression of a fibrotic disorder of the eye. Confocal and immunoelectron microscopy of the extracellular matrix of excised membranes, revealed high contents of type IV collagen. It was distributed within epiretinal membranes in basement membrane-like structures associated with cells and in interstitial deposits. In both cases, type IV collagen was always associated with type I collagen. Col IV was also coupled with Col VI and laminin. At high magnification, type IV collagen immunolabelling was associated with interstitial deposits and showed a reticular appearance due to the intersection of beaded microfilaments. The microfilaments are about 12 nm in diameter with interbead distance of 30–40 nm. Cells of the epiretinal membranes showed intracellular lysosome-like bodies heavily labeled for type IV collagen suggesting an active role in membrane remodeling. Hence, type IV collagen is not necessarily always associated with basement membranes; the molecular interactions that it may develop when not incorporated in basement membranes are still unknown. It is conceivable, however, that they might have implications in the progression of epiretinal membranes and other fibrotic disorders.

Herein biodegradable poly(e-caprolactone) was electrospun into tubular microfibrous scaffolds and modified by the surface heparinization and wrapping of Type I collagen suture for enhancing anticoa...

The purpose of this study was to combine polydioxanone and concentrated growth factor extracts to enhance the stimulation of fibroblast collagen synthesis by polydioxanone in facial plastic. In thi...

Although attempts have been made to immobilize dual short peptides on a biomaterial surface, the optimization, characterization and functional analysis of the peptide immobilization onto poly(buthy...

The aim of the study was to investigate the effect of physical training on bone parameters of rats exposed to alcohol (Al) and/or cadmium (Cd). Young female rats were divided into one control group and six groups exposed to Cd and/or Al. Al (36% calories of diet) and Cd (20 mg Cd/kg feed) were administered with liquid diet. Half of the rats from the treated groups were subjected to treadmill training (20 m/min for 0.5 h, 4 days a week). The experiment was carried out for 5 months. Al decreased the concentration of calcium (Ca) and iron (Fe) in the femur, whereas Cd and Cd + Al intake reduced the contents of Ca, Fe and zinc. Al and/or Cd caused an increase in both C-terminal telopeptide of type I collagen (CTX1; bone resorption marker) and osteocalcin (OC; formation indicator) and enhanced the degree of porosity and flexural strength of the femur. Al partially prevented the loss of Fe from the bone caused by Cd, but intensified the inhibition of growth of body weight in comparison with separate exposure to Cd. In rats co-exposed to Cd + Al, the levels of CTX1 were greater compared with those treated with Al or Cd separately, and the density was less than that in rats exposed to Al separately. The training caused increases of magnesium and Ca contents, decreases in CTX1, as well as increases in OC and bone density, decreasing their porosity. The effect of training on the bone status, however, was limited (especially in rats co-exposed to Cd and Al) because of the increase in their mineralization, stimulated by exercises, was insufficient in relation to collagen production intensity. In conclusion, training had favourable effects on some bone parameters, but did not compensate for the negative effects of Al and/or Cd exposure on the poor mineralization and histopathological and morphological changes in the femur.

Type I collagen, used as a raw material, plays a pivotal role in the development of medical devices and tissue engineering. Due to the risk of zoonotic transmission and religious constraints for mammalian collagen, fish collagen gains increased attention and is widely seen as an alternative. In this study, two collagen micro/nanofiber materials, self-assembled collagen nanofiber and electrospun collagen nanofiber, were prepared by tilapia skin collagen and their biocompatibility and immunogenicity was thoroughly investigated. The result revealed that the state of tilapia skin collagen in self-assembled collagen nanofiber and electrospun collagen nanofiber was different. The circular dichroism spectrum indicated that collagen in self-assembled collagen nanofiber retained the triple helical structure of the native collagen, while collagen in electrospun collagen nanofiber was denatured into gelatin. Nevertheless, the evaluation according to ISO10993, including tests of cytotoxicity, hemolysis, skin sensitization, acute systemic toxicity, mouse immunization and lymphocyte proliferation, demonstrated good biocompatibility and low immunogenicity for both self-assembled and electrospun collagen nanofiber materials. Overall, the present study highlighted that type I collagen from tilapia skin would be a promising biomaterial for the development of regenerate medical products.

Objective Meniscus tissue is composed of highly aligned type I collagen embedded with cartilaginous matrix. This histological feature endows mechanical properties, such as tensile strength along the direction of the collagen alignment and endurance to compressive load induced by weight bearing. The main objective of this study was to compare the fibrocartilage construction capability of different cell sources in the presence of mechanical stimuli. Design Synovial multipotent stem cells (SvMSCs) and meniscal chondrocytes (MCs) from immature and mature rabbits were maintained under similar conditions for comparative evaluation of growth characteristics and senescence tendency. The differentiation potential of cell sources, including fibrocartilage generation, were comparatively evaluated. To determine the capability of fibrocartilage generation, cultured cell sheets were rolled up to produce cable-form tissue and subjected to chondrogenic induction in the presence or absence of static tension. Results Although SvMSCs showed superior cell growth characteristics during in vitro cell expansion, senescence-associated β-galactosidase expression was consistently higher, compared with MCs. MCs showed glycosaminoglycan (GAG)-rich matrix formation during default in vitro chondrogenesis. While application of static tension significantly reduced GAG production, MCs continued to show robust tissue growth. SvMSCs showed inferior chondrogenic differentiation and diminished tissue growth in the presence of static tension. Conclusions While SvMSCs produced fibrous tissue during default in vitro chondrogenesis, their fibrocartilage generation potential in the presence of static tension was significantly lower, compared with MCs. Our results support evaluation of cellular response to tensile stimulus as a decisive factor in determining the ideal cell source for fibrocartilage reconstruction.

We have developed a unique delivery system of growth factors using collagen membranes (CMs) to induce bone regeneration. We hypothesized that fibroblast growth factor18 (FGF-18), a pleiotropic protein that stimulates proliferation in several tissues, can be a good candidate to use our delivery system for bone regeneration. Cell viability, cell proliferation, alkaline phosphatase activity, mineralization, and marker gene expression of osteoblastic differentiation were evaluated after mouse preosteoblasts were cultured with a CM containing FGF-18, a CM containing platelet-derived growth factor, or a CM alone. Furthermore, expression of microRNA, especially miR-133a and miR-135a involving inhibition of osteogenic factors, was measured in preosteoblasts with CM/FGF-18 or CM alone. A sustained release of FGF-18 from the CM was observed over 21 days. CM/FGF-18 significantly promoted cell proliferation, alkaline phosphatase activity, and mineralization compared to CM alone. Gene expression of type I collagen, runt-related transcription factor 2, osteocalcin, Smad5, and osteopontin was significantly upregulated in CM/FGF-18 compared to CM alone, and similar to CM/platelet-derived growth factor. Additionally, CM/FGF-18 downregulated expression of miR-133a and miR-135a. These results suggested that released FGF-18 from a CM promotes osteoblastic activity involved with downregulation of miR-133a and miR-135a.

Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized–solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.

Background:A variety of medications are administered to the intra-articular space for the relief of joint pain. While amide-type local anesthetics have been extensively studied, there is minimal information regarding the potential chondrotoxicity of nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid medications.Purpose:To investigate the in vitro chondrotoxicity of single-dose equivalent concentrations of ketorolac, morphine, meperidine, and fentanyl on human chondrocytes.Study Design:Controlled laboratory study.Methods:Human cartilage was arthroscopically harvested from the intercondylar notch and expanded in vitro. Gene expression of cultured chondrocytes before treatment was performed with quantitative polymerase chain reaction for type I collagen, type II collagen, aggrecan, and SOX9. Chondrocytes were then exposed to 0.01%, 0.02%, and 0.04% morphine sulfate; 0.3% and 0.6% ketorolac tromethamine; 0.5%, 1.0%, and 1.5% meperidine hydrochloride; 0.0005% and 0.001% fentanyl citrate; and saline. A custom bioreactor was used to constantly deliver medications, with the dosage of each medication and the duration of exposure based on standard dose equivalents, medication half-lives, and differences in the surface area between the 6-well plates and the native joint surface. After treatment, a live/dead assay was used to assess chondrocyte viability and if minimal cell death was detected. A subset of samples after treatment was maintained to analyze for possible delayed cell death.Results:All tested concentrations of ketorolac and meperidine caused significantly increased cell death versus the saline control, demonstrating a dose-response relationship. The morphine and fentanyl groups did not show increased chondrotoxicity compared with the saline group, even after 2 weeks of additional culture.Conclusion:In vitro exposure of chondrocytes to single-dose equivalent concentrations of either ketorolac or meperidine demonstrated significant chondrotoxicity, while exposure to morphine or fentanyl did not lead to increased cell death.

Background/aims The critical part of a bio-artificial liver device is establishment of a bioreactor filled with liver cells. However, it is still unclear how to maintain benign cell function while achieving the sufficient cell quantity. In the current study, we aim to establish a novel carrier for the culture of HepG2 cells, a liver cell line, by modifying polypropylene nonwoven fabrics with native type I collagen. Methods “Piranha” solution, KH-550 and glutaraldehyde subsequently were used to bridge native type I collagen and polypropylene nonwoven fabrics. The type I collagen-coupled polypropylene nonwoven fabric was characterized by XPS, SEM, ATR-FTIR and water contact angle measurement. Furthermore, the biocompatibility between HepG2 cells and fiber film is evaluated by the ability of cell proliferation, albumin secretion, as well as urea synthesis. Results The coating of collagen onto polypropylene fabrics was more efficient using the chemical covalent binding method than direct immersion, which was validated by the presence of collagen-related elements and chemical bond. The adding of collagen in polypropylene fabrics promoted hydrophilicity and HepG2 cell adherence. Additionally, enhanced cell proliferation, increased albumin secretion and urea synthesis were observed in HepG2 cells growing on collagen-coated polypropylene fabrics. Conclusions The collagen coated polypropylene nonwoven fabrics, acting as a feasible substrate for HepG2 cell culture, may be used as a promising liver cell carrier for artificial liver reactor.

Mutations in bone morphogenetic protein 1 (BMP1) in humans or deletion of BMP1 and related protease tolloid like 1 (TLL1) in mice lead to osteogenesis imperfecta (OI). Here, we show progressive periodontal defects in mice in which both BMP1 and TLL1 have been conditionally ablated, including malformed periodontal ligament (PDL) (recently shown to play key roles in normal alveolar bone formation), significant loss in alveolar bone mass ( P < 0.01), and a sharp reduction in cellular cementum. Molecular mechanism studies revealed a dramatic increase in the uncleaved precursor of type I collagen (procollagen I) and a reduction in dentin matrix protein 1 (DMP1), which is partially responsible for defects in extracellular matrix (ECM) formation and mineralization. We also showed a marked increase in the expression of matrix metallopeptidase 13 (MMP13) and tartrate-resistant acid phosphatase (TRAP), leading to an acceleration in periodontal breakdown. Finally, we demonstrated that systemic application of antibiotics significantly improved the alveolar bone and PDL damage of the knockdown phenotype, which are thus shown to be partially secondary to pathogen-induced inflammation. Together, identification of the novel roles of BMP1 and TLL1 in maintaining homeostasis of periodontal formation, partly via biosynthetic processing of procollagen I and DMP1, provides novel insights into key contributions of the extracellular matrix environment to periodontal homeostasis and contributes toward understanding of the pathology of periodontitis.

Sphingomyelin phosphodiesterase 3 ( Smpd3), which encodes neutral sphingomyelinase 2 (nSMase2), is a key molecule for skeletal development as well as for the cytodifferentiation of odontoblasts and alveolar bone. However, the effects of nSMase2 on the cytodifferentiation of periodontal ligament (PDL) cells are still unclear. In this study, the authors analyzed the effects of Smpd3 on the cytodifferentiation of human PDL (HPDL) cells. The authors found that Smpd3 increases the mRNA expression of calcification-related genes, such as alkaline phosphatase (ALPase), type I collagen, osteopontin, Osterix (Osx), and runt-related transcription factor (Runx)-2 in HPDL cells. In contrast, GW4869, an inhibitor of nSMase2, clearly decreased the mRNA expression of ALPase, type I collagen, and osteocalcin in HPDL cells, suggesting that Smpd3 enhances HPDL cytodifferentiation. Next, the authors used exome sequencing to evaluate the genetic variants of Smpd3 in a Japanese population with aggressive periodontitis (AgP). Among 44 unrelated subjects, the authors identified a single nucleotide polymorphism (SNP), rs145616324, in Smpd3 as a putative genetic variant for AgP among Japanese people. Moreover, Smpd3 harboring this SNP did not increase the sphingomyelinase activity or mRNA expression of ALPase, type I collagen, osteopontin, Osx, or Runx2, suggesting that this SNP inhibits Smpd3 such that it has no effect on the cytodifferentiation of HPDL cells. These data suggest that Smpd3 plays a crucial role in maintaining the homeostasis of PDL tissue.

In vivo studies have shown that the combination of infrared radiation (IR) and visible light (VIS) is responsible for the activation of metaloproteinases, causing matrix degradation and damage to healthy skin. However, the role of heat originating from the VIS spectrum on wound healing remains poorly understood. Our objective was to investigate the macroscopic, microscopic and biochemical effects of heat induced by visible light on cutaneous wound healing in mice. Male mice were anesthetized, subjected to a cutaneous excisional wound and divided into two groups ( n = 10/group) exposed to 23℃ or 43℃ in a thermal chamber for 30 min every other day, for 13 days. On day 14, the animals were sacrificed, and their lesions were processed for histochemistry, immunohistochemistry and protein expression analysis. The wound area was 42% greater 11 days ( p

The goal of this study is to understand the ability of a newly developed barrier membrane to enhance bone tissue regeneration. Here in this study we present the in vitro characterization of the barrier membrane made from type I collagen and crosslinked by oligomeric proanthocyanidins (OPCs). The effects of the membrane (P-C film) on cell cycle, proliferation, alkaline phosphatase activity, and mineralization were evaluated using the human osteoblast cell line MG-63, while the barrier ability was examined using MG-63 cells, as well as the human skin fibroblast cell line WS-1. The pore size is one of the factors that plays a key role in tissue regeneration, therefore, we evaluated the pore size of the membrane using a capillary flow porometer. Our results showed that the mean pore size of the P-C film was approximately 7–9 µm, the size known to inhibit cell migration across the membrane. The P-C film also demonstrated excellent cell viability and good biocompatibility, since the cell number increased with time, with MG-63 cells proliferating faster on the P-C film than in the cell culture flask. Furthermore, the P-C film promoted osteoblast differentiation, resulting in higher alkaline phosphatase activity and mineralization. Therefore, our results suggest that this P-C film has a great potential to be used in guided bone regeneration during periodontal regeneration and bone tissue engineering.

Background:Several sophisticated approaches to tendon engineering have been investigated as ways to improve tendon healing with the early formation of repair tissue with possibly a high amount of type I collagen. Besides the new formation of collagen type I, there is evidence for the natural integration of surrounding collagen type I from healthy tendon parts into the healing defect. However, the simple application of a type I collagen sponge to the healing site to increase the amount of local collagen type I has not been investigated.Hypothesis:Healing of the rat Achilles tendon can be accelerated by an additional supply of collagen type I, resulting in increased tear resistance.Study Design:Controlled laboratory study.Methods:The right Achilles tendons of 42 rats were transected. In half of the animals, a type I collagen sponge was placed into the gap. Animals were allowed to move freely in their cages to simulate early functional therapy. After 1, 2, and 4 weeks, tendon length, width, maximal load to failure, and stiffness were measured and the healing site studied histologically according to the Bonar score. Inflammation was evaluated by the appearance of macrophages and neutrophilic and eosinophilic granulocytes.Results:Defects receiving collagen sponges showed improved healing, with significantly stronger (29.5 vs 5.0 N, respectively, at 1 week; P = .00003), shorter (11.6 vs 14.5 mm, respectively, at 4 weeks; P = .005), thicker (10.0 vs 1.8 mm2, respectively, at 1 week; P = .00002), and less stiff (19.5 vs 30.5 N/mm, respectively, at 4 weeks; P = .02) tendons than control tendons. Overall, the biomechanical properties of the collagen-treated tendons appeared to be significantly closer to those of native, uninjured tendons compared with tendons in the control group. Histologically, no inflammatory reaction due to the collagen sponge was found.Conclusion:Tendon healing was accelerated by the type I collagen sponge. Moreover, the mechanical properties of collagen-treated tendons appeared to be significantly closer to those of normal, uninjured tendons compared with control tendons without collagen treatment.Clinical Relevance:As a simple type I collagen sponge seems to increase the amount of local collagen type I, the careful use of such sponges might be an option for tendon augmentation during Achilles tendon surgery.

Objective A meta-analysis to determine the diagnostic value of serum cross-linked N-telopeptide of type I collagen (NTx) for bone metastasis from solid tumours in the Chinese population. Methods Eligible case–control studies published up until 22 September 2014 were identified by searching the electronic literature databases PubMed®, Web of Science, China National Knowledge Infrastructure and Wanfang using the keyword ‘NTx’ in combination with ‘cancer’. A meta-analysis of the diagnostic value of serum NTx for bone metastasis from solid tumours was undertaken. Results The meta-analysis included 14 studies (1279 patients: 668 with bone metastasis; 611 controls without bone metastasis). There was a significant relationship between serum NTx concentration and bone metastasis from solid tumours in the Chinese population (odds ratio 1.39, 95% confidence intervals 1.26, 1.51). Significant heterogeneity was found in this study, but no publication bias was observed. Conclusion Serum NTx concentration may play an important role in the diagnosis of bone metastasis from solid tumours in the Chinese population.

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;WlsCO/CO mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, β-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.

Objective To characterise the effects of early and late electroacupuncture (EA) treatment on serum 17β-oestradiol (E2), C-terminal cross-linking telopeptide of type I collagen (CTX-I), bone mineral density (BMD), biomechanical bone strength and mRNA expression of cathepsin K in ovariectomised (OVX) rats. Methods Sixty Sprague-Dawley rats underwent ovariectomy (n=40) or sham surgery (n=20) and were randomly divided into two batches. Batch 1 (n=30) consisted of 10 sham-operated rats (Sham-0 group) and 20 OVX rats: half commenced EA immediately (early EA group, n=10) and half were left untreated (OVX-0 group, n=10). Batch 2 (n=30) consisted of 10 sham-operated rats (Sham-12 group) and 20 OVX rats: half commenced EA treatment 12 weeks after ovariectomy (late EA group, n=10) and half were left untreated (OVX-12 group, n=10). Rats in batches 1 and 2 were killed after 12 and 24 weeks, respectively. Serum E2, CTX-I, BMD, bone strength and cathepsin K expression were determined by ELISA, dual energy X-ray absorptiometry, biomechanical assessment and qRT-PCR, respectively. Results Both early and late EA treatment increased serum E2 levels, reduced serum CTX- I levels and increased BMD and bone strength of the L5 vertebral body in OVX rats. Although early EA treatment similarly increased BMD and bone strength of the femur, late EA treatment did not. However, both early and late EA treatment reduced mRNA expression of cathepsin K in OVX rats. Conclusions Early EA completely prevented and late EA partially prevented bone loss and deterioration of bone strength in OVX rats. The timing of initiation of EA treatment may be an important consideration for optimisation of effects. The influence of EA on bone strength appears to be at least partially mediated through regulation of the expression of cathepsin K.

Mesenchymal stem cell (MSC) transplantation by intramyocardial injection has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, low retention and survival of grafted MSCs hinder its further application. In this study, copolymer with N-isopropylacrylamide/acrylic acid/2-hydroxylethyl methacrylate-poly(ɛ-caprolactone) ratio of 88:9.6:2.4 was bioconjugated with type I collagen to construct a novel injectable thermosensitive hydrogel. The injectable and biocompatible hydrogel-mediated MSC transplantation could enhance the grafted cell survival in the myocardium, which contributed to the increased neovascularization, decreased interstitial fibrosis, and ultimately improved heart function to a significantly greater degree than regular MSC transplantation. We suggest that this novel hydrogel has the potential for future stem cell transplantation.

Subepithelial fibrosis is one of the common pathological features of asthmatic airway remodeling. During subepithelial fibrosis, type I collagen becomes the most abundant extracellular protein component. Studies have shown that Notch signaling participates in the progression of fibrosis; however, whether Notch signaling is involved in regulating type I collagen expression in airway fibroblasts remains unclear. The aim of the present study was to examine whether Notch signaling can regulate type I collagen expression in airway fibroblasts and to explore the underlying molecular mechanisms. Here, the expression of Notch signaling components was examined in mouse L929 cells and human MRC-5 cells. After upregulating or downregulating Notch signaling in these cell lines, col1α1 and col1α2 expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the role of Hes1 binding sites in both the mouse and human COL1A1 and COL1A2 promoters was investigated. This study revealed that Notch signaling-related molecules (including Notch1, Hes1, and others) are expressed in L929 and MRC-5 cells and that Notch signaling regulates the expression of col1α1 and col1α2 in both cell lines. Additionally, over-expression of the Notch intracellular domain resulted in activation of the COL1A1 and COL1A2 promoters, and site-directed mutagenesis reporter assays revealed that Hes1 proteins might augment both mouse and human COL1A1 and COL1A2 promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the COL1A1 and COL1A2 promoters in both L929 and MRC-5 cells. Therefore, it is reasonable to assume that Notch signaling can directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism, which could serve as a possible target for pharmacotherapy of airway subepithelial fibrosis.

This study analyzed the amounts of solubilized telopeptides cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and C-terminal crosslinked telopeptide of type I collagen (CTX) derived from matrix-metalloproteinases (MMPs) and cysteine cathepsins (CTPs) subsequent to application of a filler-free (Res.A) or an ion-releasing resin (Res.B) to ethylenediaminetetraacetic acid (EDTA)-demineralized dentin with or without zoledronate-containing primer (Zol-primer) pre-treatment. The chemical modification induced following treatments and artificial saliva (AS) storage was also analyzed through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Totally EDTA-demineralized specimens were infiltrated with Res.A or Res.B with or without Zol-primer pre-treatment, light-cured, and immersed in AS for up to 4 wk. ICTP release was reduced following infiltration with Res.B and further reduced when Res.B was used with Zol-primer; remarkable phosphate mineral uptake was attained after AS storage. CTX release was increased in Res.A- and Res.B-treated dentin. However, when Zol-primer was used with Res.A, the CTX release fell significantly compared to the other tested resin-infiltration methods. In conclusion, zoledronate offers an additional inhibitory effect to the ion-releasing resins in MMP-mediated collagen degradation. However, Zol-primer induces a modest reduction in CTX release only when used with resin-based systems containing no ion-releasing fillers.

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.

A previous study demonstrated that acupuncture increases the synthesis and reorganisation of collagen molecules in rat tendons after injury. Clinical studies have shown that acupuncture improves pain and functional activity in patients with tendinopathy. However, the molecular mechanisms underlying these effects are unknown. Recent studies have shown that acupuncture can modulate both anti-inflammatory (AI) and mechanotransduction (MT) molecular pathways. Moreover, the modulation of these pathways can increase type I collagen synthesis, which is the main factor that influences tendon biomechanical properties. Our hypothesis is that acupuncture increases synthesis and subsequent reorganisation of type I collagen during tendon healing by concomitant modulation of the Toll-like receptor-nuclear factor-κB AI pathway, the mitogen-activated protein kinases pathway and the Rho/Rac-F-actin MT pathway. Increased collagen synthesis and reorganisation requires that at least one acupoint is anatomically connected with the site of the injury because of the local tenoblast MT mechanism. Confirmation of this hypothesis will increase the knowledge of acupuncture modulation of the previously mentioned molecular pathways, and such confirmation may also help to establish the relationships between the different types of acupuncture needle stimulation and the influence of acupuncture stimuli on pathway activity levels. In addition, the downstream therapeutic effects of acupuncture therapy may be established. This hypothesis can be verified in a rat tendon healing model, and subsequent clinical protocols for tendon healing can be developed and evaluated as standalone therapies or as a component of a combination therapy.

Collagen from fish scale waste is currently being studied as a promising biological material to replace collagen from animals because of advantages such as safe, fat-free, not suffering from communicable diseases, and easy absorption in human body solutions. Finding the suitable process of extracting fish scale collagen is necessary because extracting collagen from fish scales by chemical methods often requires a long time. Therefore, in this paper, some bases and acids at different concentrations were chosen to find the most suitable condition for extracting fish scale collagen from the wastage of different scale fishes belonging to the familiar Cyprinus genus. The characterizations of the extracted collagen including structure, morphology, element composition, relative molecular weight, amino acid composition, denaturation temperature, crystal structure, and thermal stability were investigated. In addition, the amino acid sequence of the extracted collagen was also determined and compared with the National Center for Biotechnology Information protein database.

This study was to investigate the effects of metformin (MF) on ultraviolet A (UVA)-induced expression of matrix metalloproteinases (MMPs) and type I collagen (COL-I) in human skin fibroblasts (HSFs). HSFs were cultured in vitro and divided into control group, UVA group, and UVA + MF group. Cell proliferation was detected by CCK-8 method, and intracellular reactive oxygen species (ROS) level was detected by flow cytometry with fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) staining. Meanwhile, real-time polymerase chain reaction (PCR) was used to examine the relative messenger RNA (mRNA) expression of aging-related genes, including MMP1, MMP3, and COL-I. Moreover, the expression of MMP1, MMP3, and COL-I proteins was further detected by western blot. Compared with the control group, the ROS content in the UVA group was increased significantly ( P < 0.05), while the ROS content in the UVA + MF group was evidently lower than that in the UVA group ( P < 0.05). In addition, the MMP1 and MMP3 mRNA levels were significantly elevated, while the COL-I mRNA was significantly decreased in UVA-induced HSF cells compared with the control cells. However, MF could significantly inhibit the improved MMP1 and MMP3 mRNA level and increase the COL-I mRNA level. Moreover, MF could significantly reverse the increasing MMP1 and MMP3 protein level and decreasing COL-I protein level induced by UVA. In conclusion, MF can increase the antioxidant capacity of cells and increase the synthesis of COL-I by inhibiting the level of intracellular ROS and the expression of related MMPs, thereby inhibiting the UVA-induced photoaging effect of HSF.

The association between microfracture of the subchondral plate and a coverage scaffold has emerged as a promising strategy to treat cartilage lesions in a one-step procedure. Between different types of scaffolds (e.g. collagen, hyaluronic acid, polyglycolic acid) currently studied, type I collagen scaffold is the most used for this purpose, and is currently adopted for humans. The aim of this study was to test a novel scaffold made of mixed type I and II collagen (I-IICS) in order to define the immunological reaction of the synovial tissue and the repair capabilities induced by the collagen membrane when associated with microfracture. Eight New Zealand White rabbits, aged 180 days, were operated on bilaterally on the medial femoral condyle. A circular cartilage lesion was performed up to the calcified layer of the medial femoral condyle, and the centre of the lesion was microfractured. Randomly, one of the two lesions was covered with the I-IICS (treated), and the other was left uncovered (control). The synovial membrane reaction and the quality of the cartilage tissue repair were investigated at 2, 90, 180 and 270 days macroscopically, histomorphologically and ultrastructurally. Expression of tumor necrosis factor-alpha (TNF-α) in synovial tissue by immunocytochemistry analyses was also investigated. In the control group, at 2 days gold particles were localized mainly on synoviocyte type A, less on synoviocytes type B and on collagen bundles; in the treated group the reaction is more intense in cells in the matrix, but at 180 days controls and treated joints were very similar. The synovial membranes of the joints receiving the I-IICS did not reveal significant changes compared to the age-matched controls. Signs of inflammation were present at the 90-day time-point, and became less evident at afterwards. The degradation of the scaffolds was already evident at the 90-day time-point. The quality of the cartilage repair of the rabbits treated with the I-IICS was slightly better in 5 cases out of 6 in comparison to the controls. However, a statistically significant difference was not detected (p=0.06). Scaffolds made of mixed type I and II collagen exhibited good biocompatibility properties in vivo and favored cartilage restoration when associated with microfracture, as shown in this pilot study.

It is of critical importance to prepare three-dimensional biodegradable porous scaffolds for cartilage tissue engineering. We developed a poly(lactic- co-glycolic acid)/collagen hybrid scaffold, which combined the advantages of natural type I collagen and synthetic polymer polylactin 910 knitted mesh (90:10 copolymer of glycolic acid and lactic acid), and a method using this poly(lactic- co-glycolic acid)/collagen scaffold to regenerate cartilage with controllable shape. The mechanically strong poly(lactic- co-glycolic acid) mesh served as a mechanical skeleton supporting the scaffold, and the collagen benefited cell seeding, distribution, and tissue formation. Bovine chondrocytes were cultured in the hybrid scaffold and transplanted into the subcutaneous sites of nude mice for 4 weeks. All the samples showed spatially uniform cell distribution, natural chondrocyte morphology, and deposition of abundant cartilaginous extracellular matrices such as type II collagen and aggrecan. Production of glycosaminoglycans per DNA reached 74.63% of the natural articular cartilage. The mechanical strength was 62.76% and 71.43% in Young’s modulus and stiffness, respectively, compared to the native articular cartilage. All the samples successfully maintained the original shapes. Our method provides a new strategy for regeneration of cartilage tissue with designed shapes.

The commonly used preclinical animal model of postmenopausal osteoporosis is the mature ovariectomized rat, whereby cessation of ovarian oestrogen production consequently results in bone volume reduction. The study aim was to precisely define the time course of structural changes resulting from ovariectomy and thereby reduce the time animals have to be treated to judge the effects of osteoporosis treatment. For this purpose, we assessed architectural changes by microcomputed tomography ( μCT) during 10 weeks following ovariectomy or sham surgery at two-week intervals. Moreover, the trabecular microarchitecture of the lumbar vertebrae was assessed after necropsy. Besides this, serum biomarkers of bone turnover were determined. These data were in a new approach additionally correlated to femur mRNA expression profiles. We selected the osteoblast marker genes osteocalcin and type I collagen as well as the two osteoclast marker genes cathepsin k and tartrate-resistant acid phosphatase 5. The gene expression analysis suggested an activation of osteoblasts as well as octeoclasts. The significantly induced serum levels of osteocalcin and collagen degradation fragments also revealed this higher rate of bone turnover. Our results indicate that as soon as four weeks after ovariectomy the bone volume fraction exhibited a decline of 30% and 50% of the connectivity density. In addition, significant decreases of trabecular number and thickness as well as of the bone volume fraction were only observed in vertebrae of ovariectomized animals. Interestingly, changes of trabecular morphology were also found in the sham animals as a consequence of senescence.

Objectives: We sought to better characterize pathologic changes that occur in the human vocal fold after radiotherapy for head and neck cancer. Methods: In a blinded, controlled study of archived tissue, we evaluated postirradiation salvage laryngectomy vocal fold tissue without evidence of malignant disease. Clinical and demographic patient data were collected. In a blinded fashion, irradiated tissue was compared to nonirradiated, benign control tissue. Histomorphometric analysis was used to assess muscle and collagen organization, superficial lamina propria (SLP) and vocal ligament thickness, vocalis muscle fiber area, collagen content, and hyaluronic acid content. Immunohistochemical analysis was used to assess the content of type I collagen, type IV collagen, vimentin, fibronectin, α–smooth muscle actin, matrix metalloproteinase 9, and laminin. Results: Twenty irradiated vocal folds were evaluated and compared to control specimens. Collagen and muscle disorganization was noted in the irradiated specimens. The SLP and vocal ligament thicknesses and the mean muscle fiber diameters did not differ significantly. The SLP fibronectin and the vocalis muscle and SLP collagen content were significantly increased in the irradiated vocal folds, and the SLP collagen content increased significantly with time between irradiation and resection. The laminin content of irradiated vocalis muscles was significantly decreased. Conclusions: Radiotherapy results in significant vocal fold tissue changes. Having more precisely defined these changes, we plan continued investigation seeking targeted preventive and therapeutic interventions for improved vocal quality following radiotherapy.

Inability of chemical phosphorylation of sodium trimetaphosphate to induce intrafibrillar mineralization of type I collagen may be due to the failure to incorporate a biomimetic analog to stabilize amorphous calcium phosphates (ACP) as nanoprecursors. This study investigated adsorption/desorption characteristics of hydrolyzed and pH-adjusted sodium trimetaphosphate (HPA-Na3P3O9) to collagen. Based on those results, a 5-minute treatment time with 2.8 wt% HPA-Na3P3O9 was used in a single-layer reconstituted collagen model to confirm that both the ACP-stabilization analog and matrix phosphoprotein analog must be present for intrafibrillar mineralization. The results of that model were further validated by complete remineralization of phosphoric-acid-etched dentin treated with the matrix phosphoprotein analog and lined with a remineralizing lining composite, and with the ACP-stabilization analog supplied in simulated body fluid. An understanding of the basic processes involved in intrafibrillar mineralization of reconstituted collagen fibrils facilitates the design of novel tissue engineering materials for hard tissue repair and regeneration.

Background: The use of mesenchymal stem cells (MSCs) to treat osteochondral defects caused by sports injuries or disease is of particular interest. However, there is a lack of studies in large-animal models examining the benefits of chondrogenic predifferentiation in vitro for repair of chronic osteochondral defects. Hypothesis: Chondrogenic in vitro predifferentiation of autologous MSCs embedded in a collagen I hydrogel currently in clinical trial use for matrix-associated autologous chondrocyte transplantation facilitates the regeneration of a chronic osteochondral defect in an ovine stifle joint. Study Design: Controlled laboratory study. Methods: The optimal predifferentiation period of ovine MSCs within the type I collagen hydrogel in vitro was defined by assessment of several cellular and molecular biological parameters. For the animal study, osteochondral lesions (diameter 7 mm) were created at the medial femoral condyles of the hind legs in 10 merino sheep. To achieve a chronic defect model, implantation of the ovine MSCs/hydrogel constructs was not performed until 6 weeks after defect creation. The 40 defects were divided into 4 treatment groups: (1) chondrogenically predifferentiated ovine MSC/hydrogel constructs (preMSC-gels), (2) undifferentiated ovine MSC/hydrogel constructs (unMSC-gels), (3) cell-free collagen hydrogels (CF-gels), and (4) untreated controls (UCs). Evaluation followed after 6 months. Results: With regard to proteoglycan content, cell count, gel contraction, apoptosis, compressive properties, and progress of chondrogenic differentiation, a differentiation period of 14 days in vitro was considered optimal. After 6 months in vivo, the defects treated with preMSC-gels showed significantly better histologic scores with morphologic characteristics of hyaline cartilage such as columnarization and presence of collagen type II. Conclusion: Matrix-associated autologous chondrocyte transplantation with predifferentiated MSCs may be a promising approach for repair of focal, chronic osteochondral defects. Clinical Relevance: The results suggest an encouraging method for future treatment of focal osteochondral defects to prevent progression to osteoarthritis.

Background Despite advances in the treatment of ruptured Achilles tendon, imperfections of endogenous repair often leave patients symptomatic. Local administration of autologous conditioned serum (ACS) in patients with inflammatory, degenerative conditions has shown beneficial effects. Purpose Because ACS also contains growth factors that should accelerate tendon healing, we studied the effect of ACS on the healing of transected rat Achilles tendon. Study Design Controlled laboratory study. Methods In preliminary in vitro experiments, rat tendons were incubated with ACS and the effect on the expression of Col1A1 and Col3A1 was assessed by real-time quantitative polymerase chain reaction. To test its effect in vivo, the Achilles tendons of 80 Sprague Dawley rats were transected and sutured back together. Ten rats from each group (ACS group, n = 40; control group, n = 40) were euthanized at 1, 2, 4, and 8 weeks postoperatively for biomechanical (n = 7) and histologic (n = 3) testing. Lysyl oxidase activity was assayed by a flurometric assay. The organization of repair tissue was assessed histologically with hematoxylin and eosin- and with Sirius red-stained sections, and with immunohistochemistry. Results Tendons exposed to ACS in vitro showed a greatly enhanced expression of the Col1A1 gene. The ACS-treated tendons were thicker, had more type I collagen, and an accelerated recovery of tendon stiffness and histologic maturity of the repair tissue. However, there were no differences in the maximum load to failure between groups up to week 8, perhaps because lysyl oxidase activities were unchanged. Conclusion and Clinical Relevance Overall, our study demonstrates that treatment with ACS has the potential to improve Achilles tendon healing and should be considered as a treatment modality in man. However, as strength was not shown to be increased within the parameters of this study, the clinical importance of the observed changes in humans still needs to be defined.

After extensively expanding in monolayer culture, the cultured chondrocytes become quiescent. The aim of this study was to establish the hypothesis that the phenotypic function of extensively expanded primary chondrocytes may be restored with extracellular matrix (ECM) compositions with or without growth factors. The restoring effects of these microenvironmental factors on the near quiescent passage 9 (P9) chondrocyte were investigated. The data showed that exogenous type I collagen and type II collagen at 1:1 ratio stimulate cell proliferation greatly while type II collagen alone was enough to revive most of cartilaginous functions of near quiescent P9 chondrocytes. Exogenous type II collagen by itself was more effective in restoring cell proliferation rate, elevating glycosaminoglycan (GAG) accumulation and promoting the re-expression of type II collagen mRNAs in the near quiescent chondrocytes. The supplement of P9 chondrocytes with type II collagen plus TGF- β1 and IGF-I appeared essential for the re-expression of aggrecan and type II collagen mRNA in monolayer culture. In 3D type II collagen construct, P9 chondrocytes appeared healthy as chondrocytes and showed clear lacuna. However, in 3D type I collagen matrix, only some P9 chondrocytes exhibited lacuna. The cartilaginous microenvironments are crucial to restoring chondrocyte-phenotypic features of the quiescent or ‘dedifferentiated’ chondrocytes, implicating the potential of expanding a scarcity of healthy chondrocytes for cartilage repair or regeneration.

Polysulfone (PSU) membranes, coated and uncoated hyaluronan (HA), were compared for their ability to allow dermal fibroblast express genes related to extracellular matrix synthesis and remodeling. Fibroblasts type I and type III collagens were studied on both types of membranes; only type I collagen was synthesized on control cultures in plastic Petri dishes, whereas type III collagen was also expressed on PSU membranes. Expression of metalloproteinase (MMP)1, MMP3, and MMP2 was enhanced on PSU and HA-coated PSU membranes, with a lower level of MMP2 on HA-covered membranes. These membranes promote fetal-like matrices that provide good support for skin wound healing as well as favor nonscarring tissue repair.

Neonates with premature rupture of the membrane and oligohydramnios have an increased risk of acute respiratory morbidity. The aims of this study are to investigate the effects of experimental oligohydramnios on transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) expressions and collagen level in fetal rat lungs. On day 16 of gestation, we anesthetized timed pregnant Sprague-Dawley dams, punctured the uterine wall and fetal membranes of each amniotic sac which resulted in oligohydramnios. Fetuses in the opposite uterine horn served as controls. On days 19 and 21 of gestation, fetuses were delivered by cesarean section. Rats exposed to oligohydramnios exhibited significantly lower lung weight/body weight ratios on days 19 and 21 of gestation than did the control rats. Lung type I collagen and TGF-β1 mRNA expressions and lung collagen levels were significantly decreased in rats exposed to oligohydramnios on days 19 and 21 of gestation. Type I collagen and inhibitors of metalloproteinase-1 (TIMP-1) proteins were decreased and matrix metalloproteinase-1 (MMP-1) was increased in oligohydramnios-exposed rats on days 19 and 21 of gestation. CTGF mRNA expressions were comparable between control and oligohydramnios-exposed rats on days 19 and 21 of gestation. These data suggest that downregulation of collagen might be involved in the pathogenesis of oligohydramnios-induced respiratory morbidity.

Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67–positive epithelial cells were significantly decreased in the colonic crypts of periostin−/- mice (∼0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger (∼1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin−/- mice or periostin–non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.

Hepatic stellate cells (HSCs) are mesenchymal cells of the liver, which are normally in quiescent state and synthesize tracing amounts of extracellular matrix proteins. Upon fibrogenic stimulus, HSCs become activated and increase synthesis of type I collagen 50-100 fold. Prx1 and Prx2 are two homeobox transcription factors which are required for mesenchymal tissue formation during embryogenesis. The present study shows that Prx1 mRNA is expressed in in vivo and in vitro activated HSCs, but not in quiescent HSCs. Prx1 is also expressed in fibrotic livers, while it is undetectable in normal livers. Overexpression of Prx1a in quiescent HSCs cultured in vitro induced collagen alpha1(I) mRNA and TGFbeta3 mRNA expression. Prx1 transactivated TGFbeta3 promoter 3 fold in transient transfection experiments. In the whole liver, Prx1a induced expression of collagen alpha1(I), alpha2(I), alpha1(III) and alpha-smooth muscle mRNAs, which are the markers of activation of HSCs. Prx1 also increased expression of collagen alpha1(I) mRNA after acute liver injury. This suggests that Prx1a promotes activation of HSCs and expression of type I collagen. Several regions in the collagen alpha1(I) promoter were identified which mediate transcriptional induction by Prx1. The regions are scattered throughout the promoter and individually have modest effects; however, the cumulative effect of all sequences is50 fold. This is the first description of the effects of Prx1 in HSCs and in the liver, and identification of the two Prx1 target genes, which play a pivotal role in development of liver fibrosis, is a novel finding for liver pathophysiology.

Recent evidence suggests that uremic vascular calcification is an active, cell-mediated process resembling osteogenesis in bone rather than passive precipitation. We identified increased expression of bone-associated proteins (osteopontin, bone sialoprotein, alkaline phosphatase, type I collagen) and the bone-specific transcription factor core-binding factor α1 (Cbfα1) in histologic sections of inferior epigastric arteries obtained from patients with stage V chronic kidney disease or calcific uremic arteriolopathy. In in vitro experiments, the addition of uremic serum to cultured vascular smooth muscle cells up-regulated osteopontin and Cbfα1 expression and accelerated mineralization. This implies that the uremic mileau may lead to dedifferentiation of vascular smooth muscle cells, with subsequent mineralization. However, a lack of inhibitors of calcification may also be important. Dialysis patients with low levels of serum fetuin A, a circulating inhibitor of mineralization, have increased coronary artery calcification, and fetuin A can inhibit mineralization of vascular smooth muscle cells in vitro. Further understanding of the pathophysiology of uremic vascular calcification is needed to design effective therapeutic strategies to intervene with this devastating condition in patients with stage V chronic kidney disease.

Reconstruction of bladder and ureter tissue is indicated in cases of injury, stenosis, infection or tumor. Substitution by ileum, colon or pure synthetic polymers generates a variety of complications. Biohybrid tissue mimicking structural and functional attributes of the multilayered wall architecture of the urinary conduit may be the solution to current problems. This study reports on porcine urinary tract cells isolated and placed on UroMaix matrices with different degrees of cross-linking produced from highly purified type I collagen from medically approved porcine tissue. A patented procedure revealed membrane structures composed of a dense fibrous side and an open fibrous side. These scaffolds with the porcine urinary tract cells were incubated in a batch culture system for up to 14 days. Cell growth and topographical orientation were examined. Urothelial cells showed maximum attachment and a significant increase of living cells on the dense fiber layer of UroMaix-1. No attachment of urothelial cells occurred on the other prototypes. Smooth muscle cells showed similar behavior within the open fiber layer of all UroMaix matrices. Both urothelial and smooth muscle cells retained their phenotypes as demonstrated by the immunostaining of epithelial cytokeratin 18 and the smooth muscle myosin heavy chain respectively. Thus we could show that UroMaix scaffolds support the attachment and proliferation of urinary tract cells. The elastomeric properties of the collagenous matrices promise attractive applications in the tissue engineering of the urinary tract with its high mechanical demands.

Alpha2 integrin on fibroblasts is reported to play an important role in the induction of drug-induced gingival overgrowth, which is characterized by excessive accumulation of type I collagen in gingival connective tissue. Silent polymorphism 807 T/C within the alpha2 integrin gene is associated with high/low alpha2 integrin expression. The aim of this study was to test the hypothesis that expression of alpha2 integrin 807 T/C polymorphism correlates with drug-induced gingival overgrowth. A case-control study comparing 136 subjects taking calcium channel blockers (72 with vs. 64 without drug-induced gingival overgrowth) demonstrated that the frequency of the +807 C allele was significantly higher in the case group than in the controls (odds ratio, 3.61; 95% confidence interval, 2.14 - 6.10; P < 0.05). The present findings suggest that the alpha2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.

Osteoblast differentiation and extracellular matrix production are pivotal processes for implant osseointegration or bone tissue engineering. We hypothesized that a biomimetic coating on titanium surfaces, consisting of apatite and amelogenin, would promote such processes. Human Embryonic Palatal Mesenchymal pre-osteoblasts were used as a model for the evaluation of cell adhesion and spreading patterns, as well as mRNA expression of certain osteoblastic gene products. Real-time PCR showed significant (p < 0.05) increase in expression of type I collagen, alkaline phosphatase, and osteocalcin from cells grown on titanium with an apatite/amelogenin composite, as compared with that from cells grown on a pure titanium or apatite coating only. Osteocalcin expression was specifically stimulated by amelogenin added to the culture media. Enhanced attachment and cell spreading were also observed. The biomimetic coating promoting cell adhesion and osteoblast differentiation may have great potential for future dental and biomedical applications.