Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function.

One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage-specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three-dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D-cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage-specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region.

In vitro cytotoxicity and in vivo effects of a decellularized xenogeneic collagen scaffold in nasal cartilage repair.

Tissue engineering is considered a promising future option for nasal cartilage repair. However, until now, an optimal material has not been identified for this specific purpose. Therefore, the aim of this study was to analyze a recently developed decellularized collagen matrix, which has promising material properties for septal cartilage repair. A tetrazolium dye based cytotoxicity assay using rat nasal septum chondrocytes was performed to examine the cytotoxic effects of decellularized cartilage matrices. Unseeded scaffolds as well as scaffolds seeded with chondrocytes were implanted in nasal septum defects in Lewis rats to investigate the cellular and humoral inflammatory responses in the surrounding tissue as well as the effect on the formation of nasal septum perforations. Samples were analyzed histochemically and immunohistochemically after 1, 4, and 12 weeks. Although cells for the cytotoxicity assay were cultured under serum-free conditions for 24 h to increase sensitivity, no cytotoxic effects were detected. Histological and immunohistochemical evidence displayed that the implanted scaffolds induced minor macrophage and lymphocyte infiltration and were well integrated at the contact site to native cartilage and between the mucosal membranes. The biocompatibility index revealed only slightly irritating effects during the study period. Septal perforations were prevented efficiently. In summary, our results provide evidence that decellularized xenogeneic collagen scaffolds are suitable for cartilage tissue engineering. The scaffolds were integrated well into septal cartilage defects without causing a strong inflammatory reaction and prevented the development of nasal septum perforations. Therefore, we envision the possibility to use them in nasal cartilage repair in the future.

Prefabrication of 3D cartilage contructs: towards a tissue engineered auricle--a model tested in rabbits.

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15 × 8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

Marine collagen scaffolds for nasal cartilage repair: prevention of nasal septal perforations in a new orthotopic rat model using tissue engineering techniques.

Autologous grafts are frequently needed for nasal septum reconstruction. Because they are only available in limited amounts, there is a need for new cartilage replacement strategies. Tissue engineering based on the use of autologous chondrocytes and resorbable matrices might be a suitable option. So far, an optimal material for nasal septum reconstruction has not been identified. The aim of our study was to provide the first evaluation of marine collagen for use in nasal cartilage repair. First, we studied the suitability of marine collagen as a cartilage replacement matrix in the context of in vitro three dimensional cultures by analyzing cell migration, cytotoxicity, and extracellular matrix formation using human and rat nasal septal chondrocytes. Second, we worked toward developing a suitable orthotopic animal model for nasal septum repair, while simultaneously evaluating the biocompatibility of marine collagen. Seeded and unseeded scaffolds were transplanted into nasal septum defects in an orthotopic rat model for 1, 4, and 12 weeks. Explanted scaffolds were histologically and immunohistochemically evaluated. Scaffolds did not induce any cytotoxic reactions in vitro. Chondrocytes were able to adhere to marine collagen and produce cartilaginous matrix proteins, such as collagen type II. Treating septal cartilage defects in vivo with seeded and unseeded scaffolds led to a significant reduction in the number of nasal septum perforations compared to no replacement. In summary, we demonstrated that marine collagen matrices provide excellent properties for cartilage tissue engineering. Marine collagen scaffolds are able to prevent septal perforations in an autologous, orthotopic rat model. This newly described experimental surgical procedure is a suitable way to evaluate new scaffold materials for their applicability in the context of nasal cartilage repair.

Auricular keloids: treatment and results.

The aim of this study was to present long-term results in patients with auricular keloids after surgical excision and/or medical therapy by corticoid injection. A retrospective study at an academic tertiary referral centre is presented. Seventeen patients after excision, injection of corticoid, full skin grafting (single therapy or combination of interventions) for auricular keloids were followed up. The validated questionnaires SF-36 and patient outcomes of surgery-head/neck were applied to evaluate the quality of life and the patients' satisfaction after therapy. Photographs of the former keloid site were rated by an experienced facial plastic surgeon being unaware of treatment method and the patient's own estimation. The best results for retroauricular keloids were reached by excision, skin grafting and triamcinolone injection, and for earlobe keloids by excision, primary wound closure and triamcinolone injection. Both in rating by the patients and in grading by an investigator, the highest scores for aesthetics and satisfaction were found after triamcinolone injection together with or without excision or skin grafting. A size-related resection of keloids with defect reconstruction by full thickness skin grafting for retroauricular keloids and primary wound closure of ear lobe keloids with an additional steroid injection lead to good cosmetic results and high level of satisfaction among patients.

Hypertonic fluid resuscitation from subarachnoid hemorrhage in rats: a comparison between small volume resuscitation and mannitol.

OBJECTIVE: Death and severe morbidity after subarachnoid hemorrhage (SAH) are mainly caused by global cerebral ischemia through increased intracranial pressure (ICP) and decreased cerebral blood flow (CBF). We have recently demonstrated neuroprotective effects of small volume resuscitation (7.5% saline in combination with 6% dextran 70) in an animal model of SAH, leading to normalization of increased ICP, reduced morphological damage and improved neurological recovery. In the present study, we compared the concept of small volume resuscitation represented by two clinically licenced hypertonic-hyperoncotic saline solutions with the routinely used hyperosmotic agent-mannitol-and investigated their effects on ICP, CBF, neurological recovery and morphological damage after SAH in rats. METHODS: 60 dextran-resistant Wistar rats were subjected to SAH by an endovascular filament. ICP, MABP (mean arterial blood pressure) and bilateral local CBF were continuously recorded. All animals were randomly assigned to four groups: (I) NaCl 0.9% (4 ml/kg bw), (II) 7.5% NaCl+6% dextran 70 (4 ml/kg bw), (III) 7.2% NaCl+HES 200,000 (4 ml/kg bw) and (IV) 20% mannitol (9.33 ml/kg bw) given 30 min after SAH. Neurological deficits were assessed on days 1, 3 and 7 after SAH. The morphological damage was evaluated on day 7 after SAH. RESULTS: The induction of SAH resulted in an immediate ICP increase to 46.6+/-3.2 mm Hg (mean+/-S.E.M.) and 29.6+/-1.3 (mean+/-S.E.M.) mm Hg 90 min post-SAH. While a treatment with both hypertonic saline solutions (II, III) decreased ICP as well as the 20% mannitol solution, only the group treated with hypertonic saline and dextran 70 (II) showed an increase of ipsilateral CBF for 20 min after the infusion and significantly more surviving neurons in the motorcortex and caudoputamen. Mortality was reduced from 60% (I) and 73% (III and IV), respectively, to 40% in group II. CONCLUSION: Of all hypertonic solutions investigated, small volume resuscitation with NaCl 7.5% in combination with 6% dextran 70 evolved to be most effective in terms of reducing the initial harmful sequelae of SAH, leading to lowered ICP and less morphological damage after SAH in the rat.