A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration.

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells.

Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2 wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Biocompatibility Study Of Hydroxyapatite-Chitosan Composite For Medical Applications At Microwave Frequencies

Hydroxyapatite (HAp, Ca10(PO4)6(OH)2) bioceramic and chitosan (poly [( -1-4) D-glucosamine]) biopolymer show good biocompatibility in vivo. They have biological origin and show excellent interactions with microwave. Microwave study of HAp made using different drying techniques and their composites with chitosan in the ISM band is presented. Pastes are made using HAp and chitosan with different ratios of mixing. The dielectric properties of this composites match with that of human fat, collagen tissues. Some of the compositions exhibit dielectric property close to that of natural bone. This makes them more biocompatible and better substitutes for natural bone. Thus composite bioceramics can be considered as phantom model constituents for imaging purposes. Their dielectric properties prove that they are biocompatible.

Plastic compression of a collagen gel forms a much improved scaffold for ocular surface tissue engineering over conventional collagen gels

We compare the use of plastically compressed collagen gels to conventional collagen gels as scaffolds onto which corneal limbal epithelial cells (LECs) are seeded to construct an artificial corneal epithelium. LECs were isolated from bovine corneas (limbus) and seeded onto either conventional uncompressed or novel compressed collagen gels and grown in culture. Scanning electron microscopy (SEM) results showed that fibers within the uncompressed gel were loose and irregularly ordered, whereas the fibers within the compressed gel were densely packed and more evenly arranged. Quantitative analysis of LECs expansion across the surface of the two gels showed similar growth rates (p > 0.05). Under SEM, the LECs, expanded on uncompressed gels, showed a rough and heterogeneous morphology, whereas on the compressed gel, the cells displayed a smooth and homogeneous morphology. Transmission electron microscopy (TEM) results showed the compressed scaffold to contain collagen fibers of regular diameter and similar orientation resembling collagen fibers within the normal cornea. TEM and light microscopy also showed that cell–cell and cell–matrix attachment, stratification, and cell density were superior in LECs expanded upon compressed collagen gels. This study demonstrated that the compressed collagen gel was an excellent biomaterial scaffold highly suited to the construction of an artificial corneal epithelium and a significant improvement upon conventional collagen gels.

Photochemical cross-linking of plastically compressed collagen gel produces an optimal scaffold for corneal tissue engineering

The experiments were designed to use photochemically cross-linked plastically compressed collagen (PCPCC) gel to support corneal epithelial cells. A plastically compressed collagen (PCC) scaffold was photo cross-linked by UVA in the presence of riboflavin to form a biomaterial with optimal mechanical properties. The breaking force, rheology, surgical suture strength, transparency, ultrastructure, and cell-based biocompatibility were compared between PCPCC and PCC gels. The breaking force increased proportionally with an increased concentration of riboflavin. The stress required to reach breaking point of the PCPCC scaffolds was over two times higher compared to the stress necessary to break PCC scaffolds in the presence of 0.1% riboflavin. Rheology results indicated that the structural properties of PCC remain unaltered after UVA cross-linking. The PCC gels were more easily broken than PCPCC gels when sutured on to bovine corneas. The optical density values of PCPCC and PCC showed no significant differences (p > 0.05). SEM analyses showed that the collagen fibres within the PCPCC gels were similar in morphology to PCC gels. No difference in cell-based biocompatibility was seen between the PCPCC and PCC scaffolds in terms of their ability to support the ex vivo expansion of corneal epithelial cells or their subsequent differentiation evidenced by similar levels of cytokeratin 14. In conclusion, PCPCC scaffold is an optimal biomaterial for use in therapeutic tissue engineering of the cornea.

The bioactivity of composite Fmoc-RGDS-collagen gels

The incorporation of small bioactive peptide motifs within robust hydrogels constitutes a facile procedure to chemically functionalise cell and tissue scaffolds. In this study, a novel approach to utilise Fmoc-linked peptide amphiphiles comprising the bio-functional cell-adhesion RGDS motif within biomimetic collagen gels was developed. The composite scaffolds thus created were shown to maintain the mechanical properties of the collagen gel while presenting additional bio-activity. In particular, these materials enhanced the adhesion and proliferation of viable human corneal stromal fibroblasts by 300% compared to nonfunctionalised gels. Furthermore, the incorporation of Fmoc-RGDS nanostructures within the collagen matrix significantly suppressed gel shrinkage resulting from the contractile action of encapsulated fibroblasts once activated by serum proteins. These mechanical and biological properties demonstrate that the incorporation of peptide amphiphiles provides a suitable and easy method to circumvent specific biomaterial limitations, such as cell-derived shrinkage, for improved performance in tissue engineering and regenerative medicine applications.

Synergism Between Keratinocyte Growth Factor and Carboxymethyl Chitosan Reduces Pericardial Adhesions

Background. Mesothelial injury is the pivot in the development of adhesions. An increase in the proliferation of mesothelial cells was verified by in vitro studies with the use of keratinocyte growth factor (KGF). This study investigated the influence of KGF associated with thermo-sterilized carboxymethyl chitosan (NOCCts) in the reduction of pericardial adhesions. Methods. An induction model of pericardial adhesion was carried out in 24 pigs. Animals were randomly allocated to receive topical application of KGF, KGF + NOCCts, NOCCts, or saline (control). At 8 weeks, intra-pericardial adhesions were evaluated and a severity score was established. The time spent to dissect the adhesions and the amount of sharp dissection used, were recorded. Histologic sections were stained with sirius red for a morphometric evaluation using a computer-assisted image analysis system. Cytokeratin AE1/AE3 immunostaining were employed to identify mesothelial cells. Results. The severity score expressed in median (minimum to maximum), in relation to the control group (17 [15 to 18]), was lower in the KGF + NOCCts group (7 [6 to 9], p < 0.01) followed by the KGF group (11.5 [9 to 12], 0.01 < p < 0.05) and the NOCCts group (12 [9 to 14], p > 0.05). The dissection time was significantly lower in the KGF + NOCCts group (7.1 +/- 0.6 vs 33.9 +/- 9.2 minutes, p < 0.001). A significantly less sharp dissection was also required in the KGF + NOCCts group. In the adhesion segment, a decreased collagen proportion was found in the KGF + NOCCts group (p < 0.05). Mesothelial cells were present more extensively in groups in which KGF was delivered (p = 0.01). Conclusions. The use of KGF associated with NOCCts resulted in a synergic action that decreases postoperative pericardial adhesions in a highly significant way. (Ann Thorac Surg 2010; 90: 566-72) (C) 2010 by The Society of Thoracic Surgeons

THERMAL BEHAVIOR OF IN VITRO MINERALIZED ANIONIC COLLAGEN MATRICES

In the present study porcine skin and bovine pericardium were used as a source of type I collagen. Both were submitted to an alkaline treatment and mineralized by the alternate soaking method. Thermal stability and extent of mineralization have been investigated using DSC and TG. After alkaline hydrolysis there is a decrease in thermal stability but mineralization stabilizes collagen structure. Thermogravimetric data have shown that the amount of hydroxyapatite present in bovine pericardium matrix (45%) was greater than on porcine skin matrix (20%). Presence of hydroxyapatite was confirmed by EDX.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Different Molecular Weight Chitosan-Based Membranes for Tissue Regeneration

Natural polymers, such as chitosan, obtained from chitin, are been widely studied for use in the tissue regeneration field. This study established a protocol to attain membranes made from this biopolymer, consisting of high or low molecular weight chitosan. The biocompatibility of these membranes was histologically evaluated, comparing them to collagen membrane surgically implanted in rat subcutaneous tissue. Fifteen Holtzmann rats were divided in three experimental groups: High and Low Molecular Weight Chitosan membranes (HMWC and LMWC) and Collagen membranes (C-control group); each of them with three experimental periods: 7, 15 and 30 days. As a result, after the seven days evaluation, the membranes were present and associated with a variable degree of inflammation, and after the 15 and 30 days evaluations, the membranes were absent in all groups. It is concluded that the chitosan-based membranes were successfully attained and presented comparable resorption times to collagen membranes.

Estudos comparativos de três diferentes scaffolds pra crescimento de célula tronco mesenquimal, fibroblastos e queratinócitos

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Combining xanthan and chitosan membranes to multipotent mesenchymal stromal cells as bioactive dressings for dermo-epidermal wounds

The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

Chitosan-alginate membranes accelerate wound healing

The purpose of this study was to evaluate the efficacy of chitosan-alginate membrane to accelerate wound healing in experimental cutaneous wounds. Two wounds were performed in Wistar rats by punching (1.5 cm diameter), treated with membranes moistened with saline solution (CAM group) or with saline only (SL group). After 2, 7, 14, and 21 days of surgery, five rats of each group were euthanized and reepithelialization was evaluated. The wounds/scars were harvested for histological, flow cytometry, neutrophil infiltrate, and hydroxyproline analysis. CAM group presented higher inflammatory cells recruitment as compared to SL group on 2nd day. On the 7th day, CAM group showed higher CD11b+ level and lower of neutrophils than SL group. The CAM group presented higher CD4+ cells influx than SL group on 2nd day, but it decreased during the follow up and became lower on 14th and 21st days. Higher fibroplasia was noticed on days 7 and 14 as well as higher collagenesis on 21st in the CAM group in comparison to SL group. CAM group showed faster reepithelialization on 7th day than SL group, although similar in other days. In conclusion, chitosan-alginate membrane modulated the inflammatory phase, stimulated fibroplasia and collagenesis, accelerating wound healing process in rats.

Combining xanthan and chitosan membranes to multipotent mesenchymal stromal cells as bioactive dressings for dermo-epidermal wounds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)