[Tissue-engineered cartilage in a prefabricated microvascularized flap]. / Gezüchtetes Knorpelgewebe in einem präfabrizierten, mikrovaskulären Lappen.

INTRODUCTION: In reconstructive surgery, the integration of tissue-engineered cartilage in a prefabricated free flap may make it possible to generate flaps combining a variety of tissue components to meet the special requirements of a particular defect. The aim of the present study was to establish the technique of prefabricating a microvascular free flap by implanting a vessel loop under a skin flap in a rabbit model. The second aim was to gather experience with prelaminating the flap with autologous tissue-engineered cartilage in terms of matrix development, inflammatory reaction and host-tissue interaction. METHODS: The microvascular flap was created by implanting a vessel loop under a random pattern abdominal skin flap. The tissue-engineered cartilage constructs were made by isolating chondrocytes from auricular biopsies. Following a period of amplification, the cells were seeded onto a non-woven scaffold made of a hyaluronic acid derivative and cultivated for 2-3 weeks. One cell-biomaterial construct was placed beneath the prefabricated flap, and the others were placed subcutaneously under the abdominal skin and intermuscularly at the lower extremity. In addition, a biomaterial sample without cells was placed subcutaneously as a control. All implanted specimens were left in position for 6 or 12 weeks. After explantation, the specimens were examined by histological and immunohistological methods. The prefabricated flap was analyzed by angiography. RESULTS: The prefabricated flaps showed a well-developed network of blood vessels formed by neovascularization between the implanted vessel loop and the original random-pattern blood supply. The tissue-engineered constructs remained stable in size and showed signs of tissue similar to hyaline cartilage, as evidenced by the expression of cartilage-specific collagen type II and proteoglycans. No hints of inflammatory reactions were observed. CONCLUSION: These results show the potential of prefabricated flaps as custom-made flaps for reconstructive surgery in difficult circumstances, more or less independent of anatomical prerequisites. Cartilage tissue engineering provides a 3-dimensional structure with minimal donor-site morbidity.

[Mesenchymal stem cells--a new pathway for tissue engineering in reconstructive surgery]. / Adulte mesenchymale Stammzellen--neue Möglichkeiten der Gewebezüchtung für die plastisch-rekonstruktive Chirurgie. andreas.naumann@hno.med.uni-muenchen.de

INTRODUCTION: Mesenchymal stem cells (MSC) have the capacity to differentiate into chondrocytes with the synthesis of cartilage. This report presents the use of human adult bone marrow derived mesenchymal stem cells for tissue engineering of autologous cartilage grafts. METHODS: Human bone marrow aspirates were obtained from the iliac crest and fractionated on a Percoll gradient. The isolated hMSC were plated at 20 x 10 (6) cells per 100 mm (2) culture dish. After 21 days in culture at 37 degrees C with 5 % CO 2, the adherent multiplied MSC were trypsinized, counted, and tested for viability by trypan blue assay. The hMSCs were loaded into a sterile 15 ml polypropylene tube (0.5 Mio cells/ml) and centrifuged on the bottom of the tube at 500 g for 5 minutes. The MSC were cultivated for 3 weeks in vitro in a specific chondrogenetic medium composed of Dulbecco's Modified Eagles Medium-High Glucose supplemented with 10 ng/ml transforming growth factor-beta 1, 1 % ITS-Premix medium, 80 micro M ascorbic acid, and 100 nM dexamethasone. RESULTS: Histological and immunohistochemical studies performed after 3 weeks in three dimensional culture demonstrated the expression of cartilage specific collagen type II and X as well as proteoglycans. CONCLUSION: Human adult mesenchymal stem cells derived from bone marrow aspirates have the ability to differentiate into chondrocytes under specific culture conditions by growth factors. The use of adult mesenchymal stem cells may be a promising tool for tissue engineering of autologous cartilage grafts in reconstructive surgery in the future.

Development and clinical assessment of an artificial cornea.

Keratoprosthesis research has been a gradual, rather fragmentary process with advances being made by isolated groups of researchers. This has arisen partly because of poor funding in the area; research groups which have achieved commercial support have often had constraints upon the full disclosure of their findings. Despite these difficulties there has been real progress over the last decade by several independent groups. This article concentrates upon our own development of a hydrogel core-and-skirt keratoprosthesis, the Chirila KPro, in order to illustrate the scientific and clinical problems common to keratoprosthesis research. Pilot data from a clinical trial is presented and the priorities for future research are discussed.

The modulation of corneal keratocyte and epithelial cell responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases collagenase production in vitro.

We examined the regulation of collagenase production by rabbit keratocyte, epithelial and mixed keratocyte/epithelial cell cultures which were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies (sponge and homogeneous gels). Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response with all cell culture types. Copolymer homogeneous gels containing 2-ethoxyethyl methacrylate (EEMA) or methyl methacrylate (MMA) induced a high response in keratocyte cultures, whilst PHEMA hydrogels induced a moderate response and the phosphorylated PHEMA (phos-PHEMA) hydrogel induced no response. Epithelial cells cultured on PHEMA, copolymer and phos-PHEMA hydrogels produced less collagenase activity than the keratocyte cells. The profile of collagenases produced by epithelial cells in response to phos-PHEMA was different to that for the other hydrogels. Co-cultured cells produced higher levels of collagenase (relative to the TCP) in response to hydrogels than did either the keratocytes or epithelial cells alone, but the response of phos-PHEMA was still the lowest. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels, although this effect was less prominent in the keratocyte cultures. The markedly reduced and alternative collagenase responses to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge and morphology.

The modulation of cellular responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases macrophage collagenase production in vitro.

We examined the regulation of collagenase production by the monocyte/macrophage THP-1 cell line when these cells were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies. Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response. Copolymer hydrogels containing 2-ethoxyethyl methacrylate (EMA) or methyl methacrylate (MMA) also induced a high response, while PHEMA hydrogels induced a low level response and the phosphorylated hydrogel induced no response. This pattern was altered when the morphology of the hydrogels was changed to that of a sponge. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels. Sponges containing EMA and MMA produced low level response relative to the TCP control. PHEMA and phosphorylated sponges produced little and no response respectively. The dramatically reduced enzyme response to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge.

Clinical results of implantation of the Chirila keratoprosthesis in rabbits.

AIMS/BACKGROUND: An ideal keratoprosthesis (KPro) would closely resemble a donor corneal button in terms of its surgical handling, optics, and capacity to heal with host tissue in order to avoid many of the complications associated with the KPros which are currently in clinical use. This study was carried out to assess the long term clinical outcomes on implantation of the core and skirt poly(2-hydroxyethyl methacrylate) KPro in animals. METHODS: 20 KPros were made and implanted as full thickness corneal replacements into rabbits and followed for up to 21 months to date. RESULTS: 80% of the prostheses have been retained, with a low incidence of complications such as cataract, glaucoma, and retroprosthetic membrane formation which are frequently associated with KPro surgery. CONCLUSIONS: KPros of this type may offer promise in the treatment of patients for whom penetrating keratoplasty with donor material carries a poor prognosis. Refinement of the KPro and further animal trials, including implantation into abnormal corneas, are however mandatory before human implantation could be planned.

Assessment of anticollagenase treatments after insertion of a keratoprosthetic material in the rabbit cornea.

PURPOSE: This study was performed to evaluate the enzyme production in response to implantation of the hydrogel material used in the experimental Chirila keratoprosthesis (KPro) and to assess the effects of five topical drugs on enzyme production and activity. KPros may be extruded from the cornea as a result of tissue melting, a process that involves excessive enzyme activity. To reduce the possibility of implant loss for the hydrogel Chirila KPro, a number of antiinflammatory drugs that have been used to treat other corneal melting conditions were investigated for their effect on initial collagenase activity after the implantation of KPro material into the rabbit cornea. METHODS: Poly(2-hydroxyethyl methacrylate) sponge pieces were implanted into rabbit corneas. Prednisolone, tetracycline, medroxyprogesterone, acetylcysteine, and sodium citrate were assessed for effects on gelatinolytic activity and stromal collagenase [matrix metalloprotease-1 (MMP-1)] production in vivo and in vitro by using zymography and Western blotting techniques. RESULTS: Whereas all five anticollagenase drugs were effective in reducing gelatinolytic activity in vitro, many were ineffective in vivo. However, medroxyprogesterone caused a reduction of gelatinolytic activity in vivo. The amount of MMP-1, as measured by immunoblotting, also was reduced by medroxyprogesterone treatment when compared with untreated controls. An increase in the apparent molecular weight of MMP-1 in operated corneas appears to be the result of the association of MMP-1 with collagen fragments resulting from the surgical trauma. CONCLUSION: This study indicates that topical medroxyprogesterone may be a useful adjunctive therapy after prosthokeratoplasty.

Effect of crosslinked poly(1-vinyl-2-pyrrolidinone) gels on cell growth in static cell cultures.

Poly(1-vinyl-2-pyrrolidinone) (PVP) and copolymers of 1-vinyl-2-pyrrolidinone are insoluble in water when crosslinked but they can absorb very large amounts of water to become syringe-injectable hydrogels. Such gels have been investigated recently as potential substitutes for the vitreous humour in the eye. In this study, during the cytotoxic evaluation by sulforhodamine B colorimetric assay of variously crosslinked PVP gels, it was found that many of them showed protective/growth promoting effects on 3T3 mouse fibroblasts in static cultures, a phenomenon encountered previously only with aqueous solutions of a limited number of natural or synthetic polymers. Particularly, the gels crosslinked with diethylene glycol dimethacrylate (DEGDMA) induced a significant enhancement of cell proliferation, especially in serum-free cultures. No correlation between this effect and the essential gel properties (chemical composition, viscoelasticity and equilibrium water content) could be established. The study demonstrated that crosslinked PVP hydrogels showed a serum-like growth promoting effect on an anchorage-dependent cell line, which may be due to physical protection, inability of the insoluble gels to penetrate cell membranes, and their ability to mimic the extracellular matrix.