Intrinsic versus extrinsic vascularization in tissue engineering.
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Animals↗
Biomedical subjects
Ulrich Kneser
Publications and source records attributed to Ulrich Kneser.
Simultaneous heart valve replacement and reconstruction of the radiation-damaged chest wall with a delayed vertical rectus abdominis myocutaneous flap.
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Aged↗
Engineering of vascularized transplantable bone tissues: induction of axial vascularization in an osteoconductive matrix using an arteriovenous loop.
INTRODUCTION: Vascularization remains an obstacle to engineering of larger volume bone tissues. Our aim was to induce axial vascularization in a processed bovine cancellous bone (PBCB) matrix using an arteriovenous (AV) loop (artery, vein graft, and vein). METHODS: Custom-made PBCB discs (9 x 5 mm) were implanted into rats. In group A (n = 19), the matrices were inserted into microsurgically constructed AV loops between the femoral vessels using a vein graft from the contralateral side. In group B (n = 19), there was no vascular carrier. The matrices were encased in isolation chambers. After 2, 4, and 8 weeks, the animals were perfused with India ink via the abdominal aorta. Matrices were explanted and subjected to histological and morphometric analysis. Results were compared with intravital dynamic micro & magnetic resonance imaging and scanning electron microscopy images of vascular corrosion replicas. RESULTS: In group A, significant vascularization of the matrix had occurred by the 8th week. At this time, vascular remodeling with organization into vessels of different sizes was evident. Blood vessels originated from all 3 zones of the AV loop. Group A was significantly superior to group B in terms of vascular density and vascularization kinetics. DISCUSSION: This study demonstrates for the first time successful vascularization of solid porous matrices by means of an AV loop. Injection of osteogenic cells into axially prevascularized matrices may eventually create functional bioartificial bone tissues for reconstruction of large defects.
Animals↗
Radial collateral ligament repair of the thumb metacarpophalangeal joint using the abductor pollicis brevis tendon.
BACKGROUND: Injuries to the radial collateral ligament of the thumb metacarpophalangeal joint are less common than injuries to the ulnar side. Therefore, published data on this subject are scarce. An innovative method of radial collateral ligament repair is hereby presented, placing emphasis on the long-term results. This technically simple method was developed to allow for a biological joint stabilization based on functional and anatomical considerations. The key concept is to reestablish joint stability by spanning the tendinous portion of the insertion of the abductor pollicis brevis over the radial side of the metacarpophalangeal joint. METHODS: Nine patients with rupture of the radial collateral ligament were treated by means of the abductor pollicis brevis-plasty. All patients suffered from subacute or chronic radial ligament insufficiency. To determine the subjective results of the repair, a score was developed grading the patient's ranking of the results achieved by the abductor pollicis brevis-plasty. All patients graded the results on a scale from 1 (excellent) to 6 (unacceptable). RESULTS: Patients were operated on 37.1 +/- 14.6 days after trauma. Long-term examination was performed after 39 +/- 14.5 months. The patients showed a high satisfaction score of 1.7 +/- 0.5, with good mobility of the metacarpophalangeal I joint (range of motion, 52.2 +/- 6.7 degrees). CONCLUSION: Favorable results suggest that the abductor pollicis brevis-plasty is a suitable, safe, and technically feasible procedure for the operative treatment of chronic instability of the thumb metacarpophalangeal joint after radial hyperadduction trauma and complete rupture or deinsertion of the radial collateral ligament.
Adolescent↗
Tissue engineering of injectable muscle: three-dimensional myoblast-fibrin injection in the syngeneic rat animal model.
BACKGROUND: Surgical treatment of skeletal muscle loss resulting from trauma, tumor ablation, or inborn tissue defects is hampered by the scarcity of functional substitute tissue. By using techniques of tissue engineering, reconstitution of skeletal muscle defects might become a more viable option. However, it is necessary to develop an adequate, practical method for delivering myoblasts within a three-dimensional scaffold in vivo. The aim of this study was to create and evaluate a novel method for the transfer of myoblasts with clinically approved components within a three-dimensional matrix. METHODS: The authors injected expanded primary male myoblasts into muscle defects in female syngeneic rats using a two-way syringe (Duploject) within a three-dimensional fibrin matrix. Detection and evaluation were performed using Y chromosome in situ hybridization, antidesmin immunostaining, and hematoxylin and eosin staining. To identify possible differences by means of integration, the injected myoblasts were compared with 7 days of precultivated constructs. RESULTS: Injected myoblasts showed increasing integration into host muscle fibers in a time-dependent manner, exclusively at the injection site. Antidesmin staining revealed a conserved myogenic phenotype of transplanted cells. The fibrin matrix resolved over a period of 12 weeks, with no indication of an inflammatory reaction. No significant difference in the number of detected Y chromosome-positive nuclei was found between the two transplantation groups. CONCLUSIONS: The presented technique of myoblast-fibrin injection indicates a clinical potential for reconstruction of skeletal muscle defects in vivo using a ready-to-use device in combination with tissue-engineering methods.
Animals↗
Delayed reverse sural flap for staged reconstruction of the foot and lower leg.
BACKGROUND: Soft-tissue defects of the foot and lower leg caused by traumatic injury, tumor ablation, or infection associated with osteomyelitis often require coverage by flaps. One excellent option for reconstruction of these defects is the distally based neurofasciocutaneous sural flap. It allows rapid and reliable coverage of defects from the distal third of the lower leg to the forefoot without significant functional donor-site morbidity. However, the maximal size of the flap is limited by the delicate perfusion of the arterial network associated with the superficial sensory nerve. Delay procedures may increase the reliability of large sural flaps. METHODS: The authors successfully used delayed sural flaps based on a two-step procedure for the treatment of 11 patients (three women and eight men, age 50.1 +/- 20.0 years) with osteomyelitis (n = 3), melanoma (n = 3), sarcoma (n = 1), squamous cell carcinoma (n = 1), posttraumatic defects (n = 2), and recurrent gouty ulcer (n = 1). The delay period ranged from 7 to 15 days (9.7 +/- 3.1), the length of the flap was from 9 to 19 (14.8 +/- 3.0) cm, and the width of the flap from 7 to 12 (9.2 +/- 1.3) cm. Temporary wound coverage was achieved by vacuum-assisted closure during the delay period. RESULTS: All defects were covered successfully without major complications. CONCLUSIONS: The delay procedure positively affects the viability of large sural neurofasciocutaneous flaps. The authors recommend this modification for patients with large defects at the distal third of the lower leg or foot, requiring a two-step surgical approach due to the underlying disease.
Adult↗
Accelerated wound healing by in vivo application of keratinocytes overexpressing KGF.
Epidermal regeneration is a complex process, strongly influenced by growth factors, including keratinocyte growth factor (KGF). The objective of this study was to establish immortalized HaCaT keratinocytes and KMST-6-fibroblasts stably expressing KGF. Transfection efficiency, genomic integration, and functionality of the transgene were determined by ELISA and PCR, and KGF-expressing clones were selected using an air-liquid interface test system. HaCaT cells displayed stronger transgene expression compared to transfected fibroblasts, and the most effective HaCaT clone was incubated on a membrane carrier to form a "membrane cell graft." Twenty-one superficial second-degree burn wounds were created in each of three pigs, and wound healing capacity of the generated "polypeptide cell delivery system" after grafting was examined. Untransfected HaCaT keratinocytes and membrane-covered and untreated burn wounds served as controls. Histological and macroscopical follow-up revealed that grafting of transfected HaCaT cells resulted in complete reepithelialization within 5 days, while wounds covered with untransfected cells needed 2 days longer. At untreated sites, a thin epithelium was detectable after 10 days. The results indicate that wound healing processes can be stimulated distinctly by growth factors secreted from HaCaT cells, with a prominent role for transgenic KGF.
Animals↗
Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix.
Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.
Burns↗
Buried chip skin grafting in neuropathic diabetic foot ulcers following vacuum-assisted wound bed preparation: enhancing a classic surgical tool with novel technologies.
In patients with diabetes mellitus, complications such as polyneuropathy and peripheral angiopathy inevitably lead to diabetic foot complications including foot ulcers, gangrene, and osteoarthropathy. These conditions necessitate minor or major amputation as part of treatment. In patients with Charcot's arthropathy and predominant neuropathy, recurrent foot ulcers are common in areas of high pressure. Such high pressure is caused by the degrading of the architecture of the foot and inadequate footwear. These patients are a clinical challenge. A select group of such patients may benefit from free surgical tissue transfer, though free or local flap surgery is often difficult or even impossible owing to an impaired arterial circulation. In such wounds, surgical debridement followed by skin grafts often fail due to bacterial burden in the wounds. To circumvent these problems, the authors developed a therapeutic approach using buried chip skin grafting to close granulation wound beds in diabetic feet. Locally applied vacuum therapy (VAC) for wound bed preparation of chronic, nonresponsive foot ulcers and subsequent grafting using the burying technique with a minute fraction of skin was used. Firm closure was achieved. The closed wound was resistant to mechanical irritation.
Journal Article↗
Lysineurethanedimethacrylate--a novel generation of amino acid based monomers for bone cements and tissue repair.
A novel amino acid based dimethacrylate monomer (lysineurethanedimethacrylate, LUDM) was prepared by the addition of hydroxyethylmethacrylate (HEMA) to lysinediisocyanate (LDI). The structure was confirmed by FT-IR and 1H and 13C NMR spectroscopy as well as FAB-MS. Photopolymerized LUDM exhibited low volume shrinkage upon polymerization, good mechanical properties (Young's modulus: 3740 MPa) and high thermal stability. Osteoblast adhesion and growth on polymerized LUDM samples evidenced the biocompatibility. Further improvement of the mechanical properties was obtained by using Ca-hydroxyapatite as inorganic filler varying between 10 and 30 wt%. The Young's and flexural moduli increased with increasing filler content ranging from 3740 to 5250 MPa and from 2020 to 3690 MPa, respectively. The mechanical properties and the good biocompatibility of the lysine-based methacrylate networks make them interesting materials for medical applications, e.g. bone cements, and tissue engineering.
Biocompatible Materials↗
Pectus excavatum breast and chest deformity: indications for aesthetic plastic surgery versus thoracic surgery in a multicenter experience.
BACKGROUND: Breast and chest wall disfiguration attributable to a funnel chest is an aesthetically and sometimes functionally debilitating deformity requiring surgical correction. Whereas extensive and combined deformities of the ventral chest wall are classically corrected using a so-called minimally invasive repair of the pectus excavatum, a modified Ravitch repair, or the minimized Erlangen repair, plastic surgeons are mostly challenged with alloplastic implant corrections of mild funnel chests. The authors have introduced an endoscopic method for placement of customized implants to restore the visible and nonfunctionally disturbing deformation of mild funnel chests when only the sternal plate is involved. This study compared these different plastic surgical and thoracic surgical approaches in a multicenter experience to develop a clinical algorithm and to identify those patients not requiring bony correction but rather alloplastic endoscopic implant correction alone. METHODS: Patients with deformed rib cages and sternal plates were treated with the Erlangen minimally invasive procedure or a modified Ravitch procedure. For deformities involving the sternal bones only, endoscopically assisted minimally invasive implantation of silastic implants was performed. RESULTS: Between 1987 and 2003, 599 patients with a pectus excavatum deformity were treated surgically by the authors' group. Between 1999 and 2003, 515 patients underwent surgery using the Erlangen minimally invasive repair technique at Friedrich-Alexander University-Erlangen. In addition, 84 patients underwent surgery at the Freiburg University Medical Center. In the current series, 79 patients underwent surgery using the modified Ravitch method. The mean patient age was 20.5 years (range, 3-54 years), and the rate of postoperative relapses was 5%. The findings showed that 73% of the patients judged the aesthetic result as excellent to good, and 20% were satisfied. In contrast, only five patients were suitable for soft tissue augmentation only. Two of these patients in the initial period received custom implants presternally via classical transverse skin incisions, whereas three patients were treated with endoscopic customized implant tissue augmentation. CONCLUSION: Whereas with combined deformity of the sternal plate and the rib cage, a modified Ravitch repair yields good results, the endoscopic soft tissue correction with customized implants helps to avoid unsightly scars, allows for safe hemostasis in the dissection pocket, and leads to enhanced patient satisfaction. In the case of major chest wall deformity with orthopedic and functional relevance, a combination of the minimally invasive procedures (e.g., endoscopic correction and Erlangen repair) seems to show both optimized cosmetic results and maximized functionality.
Adolescent↗
Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.
Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.
Animals↗
Injectable liver: a novel approach using fibrin gel as a matrix for culture and intrahepatic transplantation of hepatocytes.
Cell transplantation and tissue engineering with liver cells are currently under investigation as experimental therapies for certain liver diseases. In this study we evaluated a fibrin-based gel matrix as carrier for hepatocytes in culture. Furthermore, a novel technique for direct intrahepatic injection of fibrin gel-immobilized hepatocytes was developed and evaluated in a rat model. Hepatocytes were harvested from rats. Fibrin matrix was generated with modified fibrin sealant. Cells, in medium containing epidermal growth factor and insulin, were seeded in a drop of fibrin matrix onto plastic culture dishes. Cell numbers were assessed by DNA content. Hepatocyte differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology (IH) for cytokeratin (CK)-18 and albumin. PKH26-labeled fibrin gel-immobilized hepatocytes were transplanted into liver by direct injection underneath the capsule. Fluorescence microscopy of explanted liver was performed to identify PKH26+ donor cells. Neotissue was characterized by IH for the markers CK-18, ED1, and desmin. Culture in a fibrin matrix allowed stable cell numbers and three-dimensional neotissue formation. RT-PCR and IH showed preservation of liver-specific markers CK-18 and albumin in vitro. Transplanted cells were identified by fluorescence microscopy after 2 and 7 days. CK-18 and desmin staining showed integration of hepatocytes and hepatic stellate cells into the host liver. Fibrin matrix is an appropriate environment for hepatocytes in culture. Direct intrahepatic injection of fibrin gel-immobilized hepatocytes is technically feasible. We conclude that fibrin gel immobilization is an attractive tool for the development of tissue engineering-based liver support systems.
Animals↗
Differentiation of osteoblasts in three-dimensional culture in processed cancellous bone matrix: quantitative analysis of gene expression based on real-time reverse transcription-polymerase chain reaction.
Processed bovine cancellous bone (PBCB) is an attractive material for tissue engineering of bone. It is biocompatible, osteoconductive, nonimmunogenic, and porous and its biomechanical properties are close to those of native bone. In this study, differentiation of primary rat osteoblasts (rOBs) incubated on PBCB was investigated in vitro. rOBs were isolated and expanded in two-dimensional culture. Expanded rOBs were seeded into PBCB disks and cultured either in basal medium (BM) or differentiation medium (DM) containing ascorbic acid, beta-glycerol phosphate, and dexamethasone. Alkaline phosphatase (ALP) activity and RNA expression of ALP, bone sialoprotein (BSP), collagen type I (COL1), osteocalcin (OC), and osteopontin (OPN) were assessed by chemiluminescence assay and quantitative real-time RT-PCR over 14 days. Histologic analysis was performed on day 14. ALP increased over the observation period independent of stimulation. OPN and BSP expression was significantly higher in the DM group whereas COL1 and OC expression was significantly higher in the BM group. Matrix calcification was detectable only in the DM group by von Kossa stain. The observed expression patterns suggest a physiological response of rOBs to the differentiation stimulus. PBCB is a suitable matrix for in vitro differentiation of osteoblasts. Cell-seeded PBCB is a potential osteogenic construct for in vivo application.
Alkaline Phosphatase↗
Tissue engineering of cultured skin substitutes.
Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue-engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life-saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split-thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue-engineered human skin products resembling natural human skin.
Animals↗