Dirk Johannes Schaefer

Plasmid Gene Delivery to Human Keratinocytes Through a Fibrin-Mediated Transfection System

We have developed a matrix-mediated transfection system to deliver plasmids to human keratinocytes. The matrix is a soluble, self-hardening fibrin matrix (Tissucol), Baxter) that has been used clinically. Recently it has been shown that full thickness burn wounds can be successfully treated with a keratinocyte fibrin glue suspension. Further, it has been demonstrated that hEGF transfected cells accelerate wound healing. In this study, we inoculated the matrix with the hEGF expression plasmid and resuspended the matrix with either cultured or noncultured human keratinocytes. We obtained successful transfection rates of these cells (up to a 100-fold increase compared to controls containing no EGF expression plasmid) in vitro. After transplantation to full thickness wounds on athymic mice we were able to show a 180-fold increase in EGF concentration compared to controls, which persisted over the entire 7-day monitored period, decreasing from 180 to 20 pg/mL at day seven. This unique approach indicates the possible utility to combine a matrix for cell transplantation with a transfection system to release therapeutic proteins in vitro and in vivo.

Tissue engineering in plastic reconstructive surgery

Tissue engineering (TE) is a new interdisciplinary field of applied research combining engineering and biosciences together with clinical application, mainly in surgical specialities, to develop living substitutes for tissues and organs. Tissue engineering approaches can be categorized into substitutive approaches, where the aim is the ex vivo construction of a living tissue or organ similar to a transplant, vs. histioconductive or histioinductive concepts in vivo. The main successful approaches in developing tissue substitutes to date have been progresses in the understanding of cell–cell interactions, the selection of appropriate matrices (cell–matrix interaction) and chemical signalling (cytokines, growth factors) for stimulation of cell proliferation and migration within a tissue‐engineered construct. So far virtually all mammalian cells can be cultured under specific culture conditions and in tissue specific matrices. Future progress in cell biology may permit the use of pluripotent stem cells for TE. The blueprint for tissue differentiation is the genome: for this it is reasonable to combine tissue engineering with gene therapy. The key to the progress of tissue engineering is an understanding between basic scientists, biochemical engineers, clinicians, and industry. Anat Rec 263:372–378, 2001.

Fibrin Gel-Immobilized Primary Osteoblasts in Calcium Phosphate Bone Cement: In vivo Evaluation with Regard to Application as Injectable Biological Bone Substitute

Osteogenic injectable bone substitutes may be useful for many applications. We developed a novel injectable bone substitute based on osteoblast-fibrin glue suspension and calcium phosphate bone cement (BC). Human osteoblasts were isolated from trabecular bone samples and cultured under standard conditions. Osteoblasts were suspended in fibrinogen solution (FS). BC was cured with thrombin solution. 8 × 4 mm injectable bone discs were prepared using silicon molds and a custom-made applicator device. Discs containing BC, BC/FS, or BC/FS/osteoblasts were implanted subcutaneously into athymic nude mice. After 3, 9 and 24 weeks, specimens were explanted and subjected to morphologic and biomechanical evaluation. In vitro fibrin gel-embedded osteoblasts displayed a differentiated phenotype as evidenced by alkaline phosphatase, collagen type 1 and von Kossa stains. A proportion of osteoblasts appeared morphologically intact over a 3-day in vitro period following application into the BC. BC/FS and BC/FS/osteoblast discs were sparsely infiltrated with vascularized connective tissue. There was no bone formation in implants from all groups. However, positive von Kossa staining only in BC/FS/osteoblast groups suggests engraftment of at least some of the transplanted cells. Biomechanical evaluation demonstrated initial stability of the composites. Young’s modulus and maximal load did not differ significantly in the BC/FS and BC/FS/osteoblast groups. The practicability of osteoblast-containing injectable bone could be demonstrated. The dense microstructure and the suboptimal initial vascularization of the composites may explain the lack of bone formation. Modifications with regard to enhanced osteoblast survival are mandatory for a possible application as injectable osteogenic bone replacement system.

Human Recombinant EGF Protein Delivered by a Biodegradable Cell Transplantation System

We have previously shown a new approach to expand cultured human keratinocytes and reconstitute the epidermis in full-thickness wounds using a new microsperical transport system. This was a new approach to increase the cell yield for seeding without altering the anchoring proteins by enzymatic steps. That time we used Cytodex 3 which failed to be degraded and induced an inflammatory reaction in a t-cell-deficient organism. Therefore, we have investigated another microcarrier consisting of PLGA, which is a well-known carrier material for cell culture and transplantation. After coating the PLGA carrier with gelatine the seeding time of viable cells reached 4 h and the cell gain after 7 days of spinner culture was 16-fold. At 14 days after transplantation, we could detect a new stratified epithelium in our full-thickness wound healing model. Because cytokines play a major role in wound healing, we loaded this carrier material with different concentrations of rhEGF, showing a dose dependent release of the protein in vitro and in vivo. This result might lead to a different approach in the treatment of wounds.

Der Interosseuslappen bei Weichteildefekten an Unterarm und Hand

ZusammenfassungOperationszielDeckung eines Weichteildefektes am distalen Unterarm und der Hand durch einen fasziokutanen gestielten Lappen.IndikationenMaximal 15×5 cm große primäre oder sekundäre Haut- und Weichteildefekte an der Hand oder am distalen Unterarm.KontraindikationenFehlende Arteria interossea posterior.Fehlende Anastomose der Arteria interossea posterior zur Arteria interossea anterior.Defekte von geringer Größe (unter 3×3 cm), die einen Wundverschluß durch Mobilisation des umgebenden Gewebes erlauben.OperationstechnikAnzeichnen des Lappens. Die Gefäßachse läuft in einer Linie vom Epicondylus lateralis humeri zum distalen Radioulnargelenk, wobei der Unterarm in Neutralstellung rotiert steht und der Ellenbogen 90° gebeugt ist. Die maximale Lappengröße beträgt 17 cm Länge und 7 cm Breite.Erster Hautschnitt ulnar im Bereich des angezeichneten Lappens auf die Muskelfaszie des Musculus extensor carpi ulnaris. Präparation des Septum intermusculare mit den perforierenden Hautästen der Arteria interossea posterior. Radiale Umschneidung der Hautinsel und Präparation des Septum intermusculare von radial. Achten auf die motorischen Äste des Nervus radialis. Ligieren der Arteria interossea posterior distal der motorischen Äste. Inzision der Haut über dem Gefäßverlauf, Lösen des Stieles durch Koagulation von Seitenästen zur Muskulatur und zum Knochen. Lösen des Septum intermusculare von ulnar. Darstellen der Kommunikation mit den Vasa interossea anteriora.Legen und spannungsfreies Einnähen des Lappens in den entsprechenden Defekt. Knickbildungen des Lappenstieles vermeiden. Verschluß des Hebedefektes direkt oder mit Vollhaut oder Spalthaut.ErgebnisseIn dreieihalb Jahren wurden bei zehn Patienten Weichteildefekte an der Hand mit dem dorsalen Interosseuslappen gedeckt. Die mittlere Zeit bis zum Einheilen des Lappens betrug 15,9 Tage, die mittlere Nachuntersuchungszeit 19,1 Monate. Alle Lappen sind eingeheilt. Als Komplikationen traten bei zwei Patienten vorübergehende Lappenstauungen auf.SummaryObjectivesCoverage of a soft tissue defect at the hand with a pedicled fasciocutaneous flap.IndicationsPrimary or secondary skin and soft tissue defects of the hand of less than 15×5 cm.ContraindicationsAbsent posterior interosseous artery.Absent anastomosis between posterior and anterior interosseous arteries.Defects smaller than 3×3 cm; they should be closed through mobilization of the surrounding tissues.Surgical TechniqueMarking of flap.The first skin incision is done at the ulnar side of the marked flap and deepened to the fascia of the extensor carpi ulnaris muscle. Dissection of the intermuscular septum while preserving the perforating skin vessels of posterior interosseous artery. Incision at the radial side and dissection of the intermuscular septum from radial without injuring the motor branches of the radial nerve. Ligation of the posterior interosseous artery distal to the motor branches. Detachment of the pedicle through coagulation of the branches going to muscles and bone. Detachment of the intermuscular septum from ulnar. Exposure of the communication with the anterior interosseous vessels.Spreading and tensionless suturing of flap into the defect. Avoid kinking of defect. Primary closure of the site of harvesting or coverage with full or split thickness skin graft.ResultsDuring a period of 3.5 years this technique was used in 10 patients. The average time to healing: 15.9 days. Average duration of follow-up: 19.1 months. All flaps were incorporated. The only complication was a venous congestion in the flap in 2 patients.OBJECTIVES Coverage of a soft tissue defect at the hand with a pedicled fasciocutaneous flap. INDICATIONS Primary or secondary skin and soft tissue defects of the hand of less than 15×5 cm. CONTRAINDICATIONS Absent posterior interosseous artery. Absent anastomosis between posterior and anterior interosseous arteries. Defects smaller than 3×3 cm; they should be closed through mobilization of the surrounding tissues. SURGICAL TECHNIQUE Marking of flap. The first skin incision is done at the ulnar side of the marked flap and deepened to the fascia of the extensor carpi ulnaris muscle. Dissection of the intermuscular septum while preserving the perforating skin vessels of posterior interosseous artery. Incision at the radial side and dissection of the intermuscular septum from radial without injuring the motor branches of the radial nerve. Ligation of the posterior interosseous artery distal to the motor branches. Detachment of the pedicle through coagulation of the branches going to muscles and bone. Detachment of the intermuscular septum from ulnar. Exposure of the communication with the anterior interosseous vessels. Spreading and tensionless suturing of flap into the defect. Avoid kinking of defect. Primary closure of the site of harvesting or coverage with full or split thickness skin graft. RESULTS During a period of 3.5 years this technique was used in 10 patients. The average time to healing: 15.9 days. Average duration of follow-up: 19.1 months. All flaps were incorporated. The only complication was a venous congestion in the flap in 2 patients.

The interosseous flap for coverage of soft tissue defects at the hand

ZusammenfassungOperationszielDeckung eines Weichteildefektes am distalen Unterarm und der Hand durch einen fasziokutanen gestielten Lappen.IndikationenMaximal 15×5 cm große primäre oder sekundäre Haut- und Weichteildefekte an der Hand oder am distalen Unterarm.KontraindikationenFehlende Arteria interossea posterior.Fehlende Anastomose der Arteria interossea posterior zur Arteria interossea anterior.Defekte von geringer Größe (unter 3×3 cm), die einen Wundverschluß durch Mobilisation des umgebenden Gewebes erlauben.OperationstechnikAnzeichnen des Lappens. Die Gefäßachse läuft in einer Linie vom Epicondylus lateralis humeri zum distalen Radioulnargelenk, wobei der Unterarm in Neutralstellung rotiert steht und der Ellenbogen 90° gebeugt ist. Die maximale Lappengröße beträgt 17 cm Länge und 7 cm Breite.Erster Hautschnitt ulnar im Bereich des angezeichneten Lappens auf die Muskelfaszie des Musculus extensor carpi ulnaris. Präparation des Septum intermusculare mit den perforierenden Hautästen der Arteria interossea posterior. Radiale Umschneidung der Hautinsel und Präparation des Septum intermusculare von radial. Achten auf die motorischen Äste des Nervus radialis. Ligieren der Arteria interossea posterior distal der motorischen Äste. Inzision der Haut über dem Gefäßverlauf, Lösen des Stieles durch Koagulation von Seitenästen zur Muskulatur und zum Knochen. Lösen des Septum intermusculare von ulnar. Darstellen der Kommunikation mit den Vasa interossea anteriora.Legen und spannungsfreies Einnähen des Lappens in den entsprechenden Defekt. Knickbildungen des Lappenstieles vermeiden. Verschluß des Hebedefektes direkt oder mit Vollhaut oder Spalthaut.ErgebnisseIn dreieihalb Jahren wurden bei zehn Patienten Weichteildefekte an der Hand mit dem dorsalen Interosseuslappen gedeckt. Die mittlere Zeit bis zum Einheilen des Lappens betrug 15,9 Tage, die mittlere Nachuntersuchungszeit 19,1 Monate. Alle Lappen sind eingeheilt. Als Komplikationen traten bei zwei Patienten vorübergehende Lappenstauungen auf.SummaryObjectivesCoverage of a soft tissue defect at the hand with a pedicled fasciocutaneous flap.IndicationsPrimary or secondary skin and soft tissue defects of the hand of less than 15×5 cm.ContraindicationsAbsent posterior interosseous artery.Absent anastomosis between posterior and anterior interosseous arteries.Defects smaller than 3×3 cm; they should be closed through mobilization of the surrounding tissues.Surgical TechniqueMarking of flap.The first skin incision is done at the ulnar side of the marked flap and deepened to the fascia of the extensor carpi ulnaris muscle. Dissection of the intermuscular septum while preserving the perforating skin vessels of posterior interosseous artery. Incision at the radial side and dissection of the intermuscular septum from radial without injuring the motor branches of the radial nerve. Ligation of the posterior interosseous artery distal to the motor branches. Detachment of the pedicle through coagulation of the branches going to muscles and bone. Detachment of the intermuscular septum from ulnar. Exposure of the communication with the anterior interosseous vessels.Spreading and tensionless suturing of flap into the defect. Avoid kinking of defect. Primary closure of the site of harvesting or coverage with full or split thickness skin graft.ResultsDuring a period of 3.5 years this technique was used in 10 patients. The average time to healing: 15.9 days. Average duration of follow-up: 19.1 months. All flaps were incorporated. The only complication was a venous congestion in the flap in 2 patients.OBJECTIVES Coverage of a soft tissue defect at the hand with a pedicled fasciocutaneous flap. INDICATIONS Primary or secondary skin and soft tissue defects of the hand of less than 15×5 cm. CONTRAINDICATIONS Absent posterior interosseous artery. Absent anastomosis between posterior and anterior interosseous arteries. Defects smaller than 3×3 cm; they should be closed through mobilization of the surrounding tissues. SURGICAL TECHNIQUE Marking of flap. The first skin incision is done at the ulnar side of the marked flap and deepened to the fascia of the extensor carpi ulnaris muscle. Dissection of the intermuscular septum while preserving the perforating skin vessels of posterior interosseous artery. Incision at the radial side and dissection of the intermuscular septum from radial without injuring the motor branches of the radial nerve. Ligation of the posterior interosseous artery distal to the motor branches. Detachment of the pedicle through coagulation of the branches going to muscles and bone. Detachment of the intermuscular septum from ulnar. Exposure of the communication with the anterior interosseous vessels. Spreading and tensionless suturing of flap into the defect. Avoid kinking of defect. Primary closure of the site of harvesting or coverage with full or split thickness skin graft. RESULTS During a period of 3.5 years this technique was used in 10 patients. The average time to healing: 15.9 days. Average duration of follow-up: 19.1 months. All flaps were incorporated. The only complication was a venous congestion in the flap in 2 patients.