Artificial Connective Tissue Research Articles

Highly antifouling double network hydrogel based on poly(sulfobetaine methacrylate) and sodium alginate with great toughness

Hydrogels with good antifouling and mechanical properties as well as biocompatibility have great application potential in the field of biomedicine. In this paper, a newly double network (DN) hydrogel was prepared based on zwitterionic material sulfobetaine methacrylate (SBMA) and natural polysaccharide, sodium alginate (SA). The PSBMA network is covalently crosslinked while the SA network is ionically crosslinked by Ca2+. The hybrid crosslinked double network structure endows the DN hydrogel with excellent mechanical properties (E = 0.19 ± 0.01 MPa, σ = 0.73 ± 0.03 MPa), fast self-recovery ability as well as excellent fatigue resistance. Moreover, the results show that the PSBMA/SA-Ca2+ DN hydrogel is biocompatible and resists the absorption of non-specific proteins and adhesion of microorganisms, such as cells and algae, exhibiting outstanding antifouling properties. These unique characteristics of PSBMA/SA-Ca2+ DN hydrogel make it a promising candidate for biomedical application, such as artificial connective tissues, implantable devices, and underwater equipment.

Возможность применения ионно-трековых матриц на основе полиэфиров для создания искусственной соединительной ткани

Abstract-The cultivation of Chinese hamster fibroblasts (line V79) on ion-track membranes of various polyesters has been tested. Light and electron microscopy were used to take photographs of polyester membranes with fibroblasts grown on them, which indicated the attachment and proliferation of cells on ion-track adhesive surfaces. The greatest survival of fibroblasts was observed on ion-track membranes with a pore diameter of up to 0.5 μ. It was shown that the chemical composition of the track membrane affects the survival and proliferation rate of Chinese hamster fibroblasts. Polyethylene terephtalate and polyethylene naphtalate membranes provided the highest percentage of grown colonies and the greatest proliferation rate of cells. Track-etched polyester membranes can be used to create adhesive surfaces for the cultivation of fibroblasts and the production of artificial connective tissue. Key words: ion-track membranes and technologies, tissue engineering, fibroblasts, burn therapy

Antibacterial and Antifouling Hybrid Ionic-Covalent Hydrogels with Tunable Mechanical Properties.

Because of their self-recovery ability and fatigue resistance, double-network (DN) hydrogels with hybrid ionical-covalent cross-linking have received wide attention. In this work, by a simple "one-pot" method, a novel kind of hybrid ionic-covalent chitosan/poly(sulfobetaine methacrylate) (CS/PSBMA) DN hydrogels was prepared. The hydrogels showed high tensile strength (2.0 MPa), strong elastic modulus (0.5 MPa), fast self-recovery ability as well as excellent fatigue resistance, high mechanical strength, and toughness retention rate after soaking in water for 24 h. Additionally, the mechanical properties of the DN gels were enhanced after stretch and relaxation because of the rearrangement of the CS network. More excitingly, because of the antifouling feature of PSBMA and the inherent antibacterial property of CS, the hybrid DN hydrogels demonstrated a "repel and kill" effect on microorganisms. The CS/PSBMA DN hydrogels may find potential applications in biomedical fields, such as artificial connective tissues, implantable devices, and wound dressing.

Self-Nanoemulsifying Electrospun Fiber Enhancing Drug Permeation.

Electrospun fibers are excellent drug carriers and tissue engineering scaffolds. However, approaches to promote drug permeation in tissues with such carriers remain of great interest. Here, we propose a Quality-by-Design strategy to enhance drug permeation with self-nanoemulsifying electrospun fibers. Owing to the nanoemulsion which formed spontaneously when the polymer contacts aqueous solution such as body fluid, the resulting drug-laden fibrous membrane exhibits an outstanding drug permeation and therapeutic enhancement effect in a Franz cell experiment with ex vivo abdomen skin of rats, an artificial connective tissue model, and an in vivo rheumatoid arthritis model in rats. Meanwhile, the material also shows the capacity of rational regulation on the rate of drug release. These features of the present strategy establish our material as a new efficient approach for various clinical conditions calling for cure.

Real-Time Quantification of Proteins Secreted by Artificial Connective Tissue Made from Uni- Or Multidirectional Collagen I Scaffolds and Oral Mucosa Fibroblasts

Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

In VitroandIn VivoAssessment of Oral Autologous Artificial Connective Tissue Characteristics That Influence Its Performance as a Graft

Several studies have evaluated proteins secreted by fibroblasts comprising skin substitutes, finding that they are secreted in combinations and concentrations that promote wound healing. However, assessment of proteins secreted by oral fibroblasts forming a part of oral substitutes is scarce. In our previous work, collagen type-I scaffolds (CSs) and autologous artificial connective tissue (AACT) were produced and implanted in rabbit oral lesions, evidencing that AACT outperforms CS. The present work determined the secreted factor profile of AACT in the time of grafting as well as that of the AACT embedded in the clot. It also evaluated the proliferation and viability of AACT fibroblasts to establish the dwell time of these cells in the grafted area. Finally, it assessed whether CS, AACT, and clot-embedded AACT increase fibroblast recruitment induced by a fibrin clot, because the cell migratory response has been associated with the wound-healing outcome. We found that some of the factors secreted by AACT fibroblasts are significantly different from those secreted by clot-embedded AACT fibroblasts. Also, that the profile of proteins secreted by AACT fibroblasts and clot-embedded AACT fibroblasts is different from already reported protein secretion profiles of other engineered tissues used in treating oral mucosa wounds. It was also found that AACT fibroblasts are viable when grafted and remain in the treated area for almost 2 weeks, and that the migratory response of fibroblasts to tissue-substitute stimulus is significantly less than the migratory response induced by the clot alone. Overall, data suggest that AACT secretion of proteins is modulated by three-dimensionality and environment factors. This bioactivity and the fact that AACT does not increase fibroblast migration can be held accountable for AACT's good performance as a graft.

Observation of Dynamic Strain Hardening in Polymer Nanocomposites

Most materials respond either elastically or inelastically to applied stress, while repeated loading can result in mechanical fatigue. Conversely, bones and other biomechanical tissues have the ability to strengthen when subjected to recurring elastic stress. The cyclic compressive loading of vertically aligned carbon nanotube/poly(dimethylsiloxane) nanocomposites has revealed a self-stiffening response previously unseen in synthetic materials. This behavior results in a permanent increase in stiffness that continues until the dynamic stress is removed and resumes when it is reapplied. The effect is also specific to dynamic loads, similar to the localized self-strengthening that occurs in biological structures. These observations help to elucidate the complex interactions between matrix materials and nanostructures, and control over this mechanism could lead to the development of adaptable structural materials and active, load-bearing artificial connective tissues.

Characterization of dense artificial connective tissues generated in a newly designed bioreactor

Dense connective tissues were generated simultaneously by accumulating collagen fibrils and fibroblasts on stainless steel mesh using a bioreactor system that we designed. The advantage of our system is that the artificial connective tissues can be generated within 24 hr in the absence of inhibitors against matrix metalloproteinases. The fibroblasts were suspended in 200 mL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.5 mg/mL type I collagen. The mixed solution was circulated in two types of bioreactors with cylindrical or vertical configurations to generate luminal or parenchymal tissues, respectively. The gelatin zymography showed that MMPs were first detected in the media after 8 hr from the start of circulation and reached the highest levels on day 3. Glossy white aggregates, 1–3 mm in thickness, depending on the circulation period, accumulated on mesh grids. Fibroblasts were embedded in the network of collagen fibrils and possessed oval nuclei with or without prominent cell processes to form a bipolar shape. We could not observe distended cisternae of the endoplasmic reticula, the Golgi apparatus, or exploded mitochondria, showing hypoxic degenerative alterations of fibroblasts in dense connective tissues. The artificial tissues generated by our system will be useful for biological studies and transplantation therapy.

Development and Preclinical Evaluation of Acellular Collagen Scaffolding and Autologous Artificial Connective Tissue in the Regeneration of Oral Mucosa Wounds

This work assessed wound healing response in rabbit oral lesions grafted with autologous artificial connective tissue or acellular collagen scaffolds. Autologous artificial oral connective tissue (AACT) was produced using rabbit fibroblasts and collagen I scaffolds. Before implantation, AACT grafts were assayed to demonstrate the presence of fibroblasts and extracellular matrix components, as well as the expression of characteristic genes and secretion of chemokines, cytokines, and growth factors. AACT grafts were tested in the rabbits from which the fibroblasts were obtained, whereas acellular collagen type I scaffolds (CS) were evaluated in a separate group of rabbits. In both cases, contralateral wounds closed by secondary intention were used as controls. In a separate experiment, AACT-grafted wounds were directly compared with contralateral CS-grafted wounds in the same animals. Wound contraction and histological parameters were examined to evaluate closure differences between the treatments in the three animal experiments performed. Contraction of wounds grafted with AACT and CS was significantly lower than in their controls (p < 0.05). Additionally, AACT significantly lowered wound contraction when compared with CS (p < 0.05). Intriguingly, it was observed that AACT-grafted wounds initially displayed a significantly higher (p < 0.05)-albeit transient-inflammatory response than seen in CS-grafted wounds and secondary healed wounds. This suggests that an early inflammatory component may contribute to tissue regeneration. Altogether, the results suggest that AACT- and CS-grafted wounds favor regeneration of oral mucosa.

P163 Recapitulating the features of mild, moderate and severe dysplasia using keratinocytes derived from clinical lesions and artificial connective tissue supports

P163 Recapitulating the features of mild, moderate and severe dysplasia using keratinocytes derived from clinical lesions and artificial connective tissue supports

Prevention of anastomosis leakage: An artificial connective tissue

Anastomosis leakage still causes considerable morbidity and mortality after digestive tract surgery. We report here our experience of the use of reconstituted connective tissue created from elastin, fibronectin and collagen in preventing anastomosis leakage and closing intestinal fistulas. In animal studies (56 rats), this connective tissue was used as a patch applied with fibrin sealant to the edges of a 1 cm diameter colonic defect. In a clinical evaluation, eight patients had an intestinal fistula closed by a simple sewn suture reinforced by a sealed patch and seven patients had a high risk anastomosis reinforced by a sealed patch, in most cases as an alternative to staged surgery. In animal studies, complete reconstitution of the three colonic layers was obtained without retraction and without inflammatory reaction within 40 days, while the patch was slowly resorbed. In clinical trials all 15 defects and anastomoses healed without complication. Three patients died from other causes and, at autopsy, the patch was found to have remained in place covering the suture line. If these results are confirmed by a prospective clinical trial, such biomaterial offers the possibility of reducing the occurrence of anastomosis leakage, especially in high risk circumstances.

Viscoelastic behavior of composite ligament prostheses.

Despite the compelling need for artificial connective tissue replacements for orthopedic applications, to date, there is no material which can adequately reproduce the mechanical behavior of natural tissue with necessary long-term endurance. In this work, we introduce a novel soft composite material as a more suitable candidate for connective tissue replacement. The material proposed is based on a hydrogel-polymer matrix reinforced with poly(ethylene terephthalate) fibers wound helically to mimic the architecture of the collagen fibers in natural tissue. Macroscopic behaviors such as static stress-strain, stress relaxation, and dynamic frequency responses can be modulated with choice of the components and design of the composite structure. In doing so, the mechanical characteristics of natural ligaments can be qualitatively reproduced and sustained over time.

Artificial Tissues in Perfusion Culture

In the stagnant environment of traditional culture dishes it is difficult to generate long term experiments or artificial tissues from human cells. For this reason a perfusion culture system with a stable supply of nutrients was developed. Human chondrocytes were seeded three-dimensionally in resorbable polymer fleeces. The cell-polymer tissues were then mounted in newly developed containers (W.W. Minuth et al, Biotechniques, 1996) and continuously perfused by fresh medium for 40 days. Samples from the effluate were analyzed daily, and the pH of the medium and glucose concentration remained stable during this period. The lactid acid concentration increased from 0.17 mg/ml to 0.35 mg/ml, which was influenced by the degradation of the resorbable polymer fibers used as three dimensional support material for the cells. This perfusion system proved to be reliable especially in long term cultures. Any components in the culture medium of the cells could be monitored without disturbances as caused by manual medium replacement. These results suggest the described perfusion culture system to be a valuable and convenient tool for many applications in tissue engineering, especially in the generation of artificial connective tissue.

In vitro resistance of artificial connective tissues to human bile and pancreatic juice

The resistance of connective tissue generated from elastin, placenta extracts and human collagen was tested by in vitro incubation with bile and pancreatic juice to investigate the potential use in human digestive surgery. The resistance, determined by macroscopical and microscopical examinations, has been directly compared to those of bovine collagen matrices (Spongel®, Pangen®, Helistat®, Translagen®). Only the compact and cross-linked human collagen patch resisted both bile and pancreatic juice. All other biomaterials tested dissolved either in bile or in pancreatic juice. The in vivo behaviour of this new human collagen matrix and its involvement in gastrointestinal wound healing must now be investigated.

A new reconstituted connective tissue matrix: preparation, biochemical, structural and mechanical studies.

A fibrinogen derivative generated by thrombin was reacted with elastin to yield a new addition product or adduct between the two proteins. Addition of fibronectin, and then of collagen, did not interfere with the basic elastin-fibrinogen reaction and conferred the qualities of an artificial connective tissue to the product. Biochemical, structural and biomechanical aspects of the new matrix were studied. Aprotinin, heparin, thiomersal, and thiourea did not inhibit the main reaction; indeed, some of these ingredients improved the matrix cohesion. Scanning electron microscopy showed the genesis of a true network whose meshes were more reticulated by the addition of thiourea. Biomechanical studies, i.e., strength and elasticity showed the thiourea matrix to be the strongest. These intrinsic properties suggest the product could have biological and clinical applications.

肉芽嚢及び人工結合織嚢の吸収能に関する研究

肉芽嚢及び人工結合織嚢の吸収能に関する研究