Charles J. Doillon

The stimulation of angiogenesis and collagen deposition by copper.

Copper is known to trigger endothelial cells towards angiogenesis. Different approaches have been investigated to develop vascularisation in biomaterials. The angiogenic and healing potential of copper ions in combination with two major angiogenic factors was examined. A 3D culture system in which, under stimulation by FGF-2 and to a lesser degree with VEGF, endothelial cells assembled into structures resembling to an angiogenic process was used. The combination of CuSO(4) with increasing doses of VEGF or FGF-2 enhanced the complexity of angiogenic networks in a significant manner. In vivo studies were also conducted by incorporating FGF-2 with CuSO(4) in a cylindrical collagen-based scaffold. CuSO(4) enhanced significantly the invasion of microvessel compared to control implants and to 20ng FGF-2+/-CuSO(4). Vascular infiltration was also significantly improved by combination of CuSO(4) with FGF-2, compared to FGF-2 alone (0.2 and 1microg). Nevertheless, in comparison with CuSO(4) alone, there was a significant increase only with 1microg of FGF-2 combined with CuSO(4). Significantly, collagen fiber deposition was enhanced following the combinatory loading in comparison to that with FGF-2 alone but not with CuSO(4) only. Thus, copper associated with growth factors may have synergistic effects which are highly attractive in the fields of tissue engineering (e.g., bone) and biomaterials.

Angiogenesis in Calcium Phosphate Scaffolds by Inorganic Copper Ion Release

Angiogenesis in a tissue-engineered device may be induced by incorporating growth factors (e.g., vascular endothelial growth factor [VEGF]), genetically modified cells, and=or vascular cells. It represents an important process during the formation and repair of tissue and is essential for nourishment and supply of reparative and immunological cells. Inorganic angiogenic factors, such as copper ions, are therefore of interest in the fields of regenerative medicine and tissue engineering due to their low cost, higher stability, and potentially greater safety compared with recombinant proteins or genetic engineering approaches. The purpose of this study was to compare tissue responses to 3D printed macroporous bioceramic scaffolds implanted in mice that had been loaded with either VEGF or copper sulfate. These factors were spatially localized at the end of a single macropore some 7 mm from the surface of the scaffold. Controls without angiogenic factors exhibited only poor tissue growth within the blocks; in contrast, low doses of copper sulfate led to the formation of microvessels oriented along the macropore axis. Further, wound tissue ingrowth was particularly sensitive to the quantity of copper sulfate and was enhanced at specific concentrations or in combination with VEGF. The potential to accelerate and guide angiogenesis and wound healing by copper ion release without the expense of inductive protein(s) is highly attractive in the area of tissue-engineered bone and offers significant future potential in the field of regenerative biomaterials.

Artificial human corneas: Scaffolds for transplantation and host regeneration

Purpose To review the development of artificial corneas (pros-theses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. Methods Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. Results Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. Conclusions Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

Heparin-fibroblast growth factor-fibrin complex : in vitro and in vivo applications to collagen-based materials

Biological molecules such as fibrin and growth factors could have interesting features to design bioactive biomaterials and particularly collagen-based materials used as connective tissue replacement. Different combinations of fibroblast growth factor (FGF) and heparin complexed to fibrin were analysed. In vitro, FGF bound to matrix was rapidly, but partially released, specifically with heparin. Heparin concentrations were progressively equilibrated between matrix and medium. DNA replication of fibroblasts grown either on or within fibrin matrices was increased in the presence of both FGF and high doses of heparin incorporated in fibrin. Subcutaneous implantations of collagen sponges impregnated with composite fibrin matrices showed qualitative and quantitative tissue ingrowth within the sponges. The uncross-linked collagen of fibrin-impregnated sponges swelled after implantation. The resulting fibroblast-infiltrated tissue resembled a normal dense connective tissue that was observed particularly in the presence of high doses of heparin and FGF incorporated in fibrin.

Brushite–collagen composites for bone regeneration

Brushite-based biomaterials are of special interest in bone regeneration due to their biocompatibility and biodegradability; on the other hand, collagen is a well-known osteoconductive biomaterial. In the present study a new brushite-collagen composite biomaterial is reported. This new biomaterial was prepared by combining citric acid/collagen type I solutions with a brushite cement powder. The obtained biomaterial was a cement paste, with improved handling properties. The effect of collagen on the setting reaction of brushite cement was studied, and was found to speed up the cement setting reaction. The cement paste set into a hard ceramic material within 18.5+/-2.1min and had compressive strength similar to that of spongeous bone (48.9+/-5.9MPa in dry conditions and 12.7+/-1.5MPa in humid conditions). The combination of collagen with citric acid revealed an interesting synergistic effect on the compressive strength of the composite material. Moreover, this new biomaterial had excellent cohesion properties (ninefold better than brushite cement), and high cellular adhesion capacity (threefold higher than brushite cement). The composite biomaterial described in this study combines good handling properties, compressive strength, cohesion and cell adhesion capacity, along with the osteoconductive and biodegradable properties inherent in brushite and in collagen-based biomaterials.

Osteoblast-derived survival factors protect PC-3 human prostate cancer cells from adriamycin apoptosis

OBJECTIVES Hormone-independent and cytotoxic drug-resistant tumor growth in osteoblastic metastases defines poor survival in patients with advanced prostate cancer. Therefore, we analyzed the ability of human osteoblast-like cells (MG-63 cells) and MG-63 conditioned media (MG-63 CM) to protect PC-3 human prostate cancer cells from adriamycin cytotoxicity in vitro. METHODS Adriamycin cytotoxicity was assessed in MG-63 osteoblast-like and PC-3 prostate cancer monolayer and three-dimensional collagen coculture systems using the DNA content and trypan blue exclusion assays, analysis of indexes of cell cycle by flow cytometry, determination of DNA fragmentation on simple agarose gel and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and immunocytochemistry. RESULTS Adriamycin (100 nM) arrested both the PC-3 and MG-63 cells at the G2/M phase in the cell cycle but induced apoptosis only in PC-3 cells, as assessed by flow cytometry, trypan blue exclusion, and agarose gel. Optimal doses of MG-63 CM (50 microg/mL), insulin-like growth factor I (50 ng/mL), and transforming growth factor-beta-1 (25 ng/mL), as determined by DNA content assay, partially neutralized the adriamycin cytotoxicity of PC-3 cells detected by flow cytometry and trypan blue exclusion. In addition, MG-63 cells rescued PC-3 cells from adriamycin apoptosis in the three-dimensional type I collagen gel coculture system, as analyzed by TUNEL assay. CONCLUSIONS These data suggest that osteoblast-like cells and osteoblast-derived growth factors can optimize survival of metastatic prostate cancer cells, thereby helping to develop cytotoxic drug-resistant growth in vitro.

Collagen Biomineralization In Vivo by Sustained Release of Inorganic Phosphate Ions

A new strategy for mineralized tissue formation in vivo is presented based on localized sustained delivery of inorganic orthophosphate (Pi) sufficient to supersaturate tissue surrounding an implant and induce mineralization of collagen. After 15 days implantation mineral formation around the implants was detected. Histology and electron microscopy show two populations of apatite; inter-fibrillar microcrystals and nanocrystals associated with collagen.

Biological molecule-impregnated polyester : an in vivo angiogenesis study

Specific extracellular matrix molecules and growth factors (GFs) with angiogenic properties could be combined with biomaterials to enhance angiogenesis and subsequently tissue ingrowth through the wall of the porous structure. In this study, composite fibrin matrices containing hyaluronic acid (HA), fibronectin (FN) and/or fibroblast growth factor-1 (FGF-1), FGF-2 and an endothelial cell growth supplement (ECGS) were adsorbed onto Dacron meshes which were then implanted subcutaneously in mice. The release from the implants and the tissue distribution of implanted GFs were determined in vivo using radiolabelled FGF-2. Angiogenesis was quantified by counting the number of capillaries present in each Dacron histological serial section. Radiolabelled GF was rapidly released from matrices and was absent from them by day 28. A very low percentage of the implanted radiolabelled GFs was found in the kidneys and livers of the animals. The number of microvessels formed within fibrin-impregnated samples was increased in the presence of HA and ECGS at 14 d and of FN and ECGS or FGF-2 at 28 d. FGF-1 had no direct effect on angiogenesis in our model. These results indicate that enhancement of vascularization within prosthesis mesh may be achieved by using fibrin as a support for angiogenic molecules such as HA, FN and FGFs.

Porosity and biological properties of polyethylene glycol-conjugated collagen materials.

Collagen-based materials can be designed for use as scaffolds for connective tissue reconstruction. The goal of the present study was to evaluate the behavior of collagen materials as well as cell and tissue reactions after the conjugation of activated polyethylene glycols (PEGs) with collagen. It is known that proteins conjugated with PEGs exhibit a decrease in their biodegradation rate and their immunogenicity. Different concentrations and molecular weights of activated PEGs (PEG-750 and PEG-5000) were conjugated to collagen materials (films or sponges) which were then investigated by collagenase assay, fibroblast cell culture, and subcutaneous implantation. PEG-conjugated collagen sponge degradation by collagenase was delayed in comparison to untreated sponges. In culture, fibroblasts with a normal morphology reached confluency on PEG-conjugated collagen films. In vivo, the porous structure of non-modified sponges collapsed by day 15 with a few observable fibroblasts between the collagen fibers. In PEG-modified collagen sponges, the porous structure remained stable for 30 days. Cell infiltration was particularly enhanced in PEG-750-conjugated collagen sponges. In conclusion, PEGs conjugated onto collagen sponges stabilize the porous structure without deactivating the biological properties of collagen. These porous composite materials could function as a scaffold to organize tissue ingrowth.

Collagen-poly(N-isopropylacrylamide)-based membranes for corneal stroma scaffolds.

Purpose To investigate the feasibility of using the biocompatibility of collagen-based blended biomaterials as cell-delivery systems in ocular surface reconstruction in vivo. Methods Collagen-based composites that were blended with synthetic acrylamide-based polymers [poly(N-isopropylacrylamide), pNIPAAm] were transplanted into corneal pockets of white rabbits, with a 3-mm epithelial window. Epithelial cells were allowed to migrate onto the polymer. Transplanted eyes were examined daily for up to 30 days, after which animals were killed for histologic examination. Immunohistochemistry was performed for vimentin, &agr;–smooth muscle actin (&agr;-SMA), CD4, and CD8. Gold-chloride staining was performed to observe neuronal regrowth. Human amniotic membranes (AMs) and sham-operated corneas served as controls. All animals received topical antibiotics (levofloxacin) without the use of steroids or other immunosuppressive agents. Results The pNIPAAm polymer allowed smooth epithelialization of the cornea, which was similar to the epithelialization observed in sham controls and AM-transplanted eyes. Histology revealed that epithelium overlying the polymer was bundled into several layers, without the orientation observed with AM and sham controls. The polymer gradually thinned and was gradually replaced by host tissue. Vimentin- and &agr;-SMA–positive cells were found in stromal pockets up to 1 month following polymer transplantation. These cells were responsible for slight subepithelial haze near the wound edge. CD4- and CD8-positive lymphocytes were also observed in the vicinity of the polymer. Gold-chloride staining showed nerve regrowth in the wound edge after 1 month and subepithelial branches after 3 months. Conclusion Collagen–pNIPAAm blended polymers may be effective as biomaterials to be used in the early stages of lamellar stromal replacement.