In vitro construction of artificial blood vessels using spider silk as a supporting matrix.

Recent research has vigorously pursued the development of tissue-engineered vascular grafts (TEVs). One of the striking points of the early phase is the instability of the artificial blood vessels and lack of vascular resistance, which is supposed to be a consequence of unstable scaffolds. Therefore, alternative biological approaches are necessary to improve the physical properties of the artificial vessel walls. We developed blood vessel-like constructs based on native spider silk as a scaffold. C2C12 and ST1.6R cells were seeded on the surface of scaffolds and cultivated under pulsatile flow (max. ~135  mmHg and min. ~90  mmHg) in a bioreactor. Constructed grafts were compared to human blood vessels and cell seeded scaffolds cultured in the absence of bioreactor. Mechanical properties, morphological structures and expression of marker genes were assessed by strength and strain experiments, SEM, histological staining, immunohistology, Western blotting and quantitative real-time PCR. The results indicate that the constructed vessel resembles native blood vessels in morphological structure as well as in function and expression of biomarkers. Spider silk scaffolds seem to provide an optimal and stable basis for vessel constructs.

[Regenerative therapy approaches in plastic surgery]. / Regenerative Therapieansätze in der plastischen Chirurgie.

BACKGROUND: The regeneration of tissue lost due to trauma or disease is considered as being ideal for reconstruction with respect to repair in which a donor defect arises in a different part of the body. Through a better understanding of the cellular and molecular mechanisms of healing, possibilities for regenerative therapies have been developed in recent years. OBJECTIVES: To give an exemplary representation of current regenerative therapy approaches and their effect and clinical application. MATERIAL AND METHODS: The PubMed database was searched for different regenerative approaches in plastic surgery and various methods are presented in this article. RESULTS: Cell-based approaches, in which autologous mesenchymal stem cells from adipose tissue are preferably used, led to excellent healing results with minimal donor site morbidity. Likewise, growth factor-based approaches or the use of platelet-rich plasma achieve very good results in the field of wound and bone healing. DISCUSSION: By using different cells or molecules and thus taking advantage of biological mechanisms, the regenerative capabilities of adult organisms could be improved. Many methods have already been implemented in clinical practice, not only in reconstructive but also in aesthetic surgery. However, the success should not conceal the potential risk that is inherent in both cell and growth factor-based approaches. Until long-term experiences of such therapies have been acquired, they should be used cautiously.

Microneedling: matching the results of medical needling and repetitive treatments to maximize potential for skin regeneration.

The benefits and risks of singular and repetitive microneedling (1 mm) have not been thoroughly investigated. The aim of this study was to evaluate the benefits and risks of singular and repetitive skin needling with a microneedling device in an animal model with and without skincare. 30 Sprague Dawley rats were randomized to five groups: control, skin-care only (Vitamin A & C), 1× needling 1 mm, 4× needling 1 mm, 4× needling 1 mm with skin-care. All animals were euthanized after 10 weeks. Skin specimens were stained with HE and Masson's trichrome. Additionally, gene expression analysis with microarray technique for various growth factors (TGFß1-3, FGF, EGF, VEGF, TNF-α) and real time reverse transcription PCR for collagen I & III were conducted. We showed that singular microneedling matches and repetitive microneedling sessions superposition epidermal and dermal benefits such as an increase of epidermal thickness (up to 658% increase, p value 0.0008) and dermal connective tissue--even more so when combined with skin-care with vitamin A and C. Juvenile collagen I showed itself up-regulated in all groups, while collagen III was down-regulated. Singular and repetitive PCI with a microneedling device can achieve and supersede the results already shown with medical needling.

Inflammatory response to burn trauma: nicotine attenuates proinflammatory cytokine levels.

OBJECTIVE: The immune response to an inflammatory stimulus is balanced and orchestrated by stimulatory and inhibitory factors. After a thermal trauma, this balance is disturbed and an excessive immune reaction with increased production and release of proinflammatory cytokines results. The nicotine-stimulated anti-inflammatory reflex offsets this. The goal of this study was to verify that transdermal administration of nicotine downregulates proinflammatory cytokine release after burn trauma. METHODS: A 30% total body surface area full-thickness rat burn model was used in Sprague Dawley rats (n = 35, male). The experimental animals were divided into a control group, a burn trauma group, a burn trauma group with additional nicotine treatment, and a sham + nicotine group with 5 experimental animals per group. The last 2 groups received a transdermal nicotine administration of 1.75 mg. The concentrations of tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 were determined in homogenates of hearts, livers, and spleens 12 or 24 hours after burn trauma. RESULTS: Experimental burn trauma resulted in a significant increase in cytokine levels in hearts, livers, and spleens. Nicotine treatment led to a decrease of the effect of the burn trauma with significantly lower concentrations of tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 compared to the trauma group. CONCLUSIONS: This study confirms in a standardized burn model that stimulation of the nicotinic acetylcholine receptor is involved in the regulation of effectory molecules of the immune response. Looking at the results of our study, further experiments designed to explore and evaluate the potency and mechanisms of the immunomodulating effects of this receptor system are warranted.

Sural nerve defects after nerve biopsy or nerve transfer as a sensory regeneration model for peripheral nerve conduit implantation.

Nerve repair after injury can be effectively accomplished by direct suture approximation of the proximal and distal segments. This is more successful if coadaptation can be achieved without tension. Currently, the gold standard repair of larger deficits is the transplantation of an autologous sensory sural nerve graft. However, a significant disadvantage of this technique is the inevitable donor morbidity (sensory loss, neuroma and scar formation) after harvesting of the sural nerve. Moreover, limitation of autologous donor nerve length and fixed diameter of the available sural nerve are major drawbacks of current autograft treatment. Another approach that was introduced for nerve repair is the implantation of alloplastic nerve tubes made of, for example, poly-L-lactide. In these, nerve stumps of the transected nerves are surgically bridged using the biosynthetic conduit. A number of experimental studies, primarily in rodents, indicate axonal regeneration and remyelination after implantation of various conduits. However, only limited clinical studies with conduit implantation have been performed in acute peripheral nerve injuries particularly on digital nerves. Clinical transfer of animal studies, which can be carefully calibrated for site and extent of injury, to humans is difficult to interpret due to the intrinsic variability in human nerve injuries. This prevents effective quantification of improvement and induces bias in the study. Therefore, standardization of lesion/repair in human studies is warranted. Here we propose to use sural nerve defects, induced due to nerve graft harvesting or from diagnostic nerve biopsies as a model site to enable standardization of nerve conduit implantation. This would help better with the characterization of the implants and its effectiveness in axonal regeneration and remyelination. Nerve regeneration can be assessed, for example, by recovery of sensation, measured non-invasively by threshold to von Frey filaments and cold allodynia. Moreover, the implantation of nerve conduits may not only serve as a model to examine nerve repair, but it could also prevent neuroma formation, which is a major morbidity of sural nerve extraction.

Percutaneous collagen induction-regeneration in place of cicatrisation?

BACKGROUND: Ablative procedures that are used for the improvement of a degenerative process that leads to a loss of skin elasticity and integrity, injure or destroy the epidermis and its basement membrane and lead to fibrosis of the papillary dermis. It was recently shown in clinical and laboratory trials that percutaneous collagen induction (PCI) by multiple needle application is a method for safely treating wrinkles and scars and smoothening the skin without the risk of dyspigmentation. In our study, we describe the effect of PCI on epidermal thickness and the induction of genes relevant for regenerative processes in the skin in a small animal model. METHODS: The purpose of this study in a rat model was to determine the effects of PCI on the skin both qualitatively and quantitatively. The epidermal and dermal changes were observed by histology and immunofluorescence. The changes in gene expression were measured by array analysis for cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-7, epidermal growth factor (EGF) and extracellular matrix molecules such as collagen type I and type III. RESULTS: The present study showed that PCI with topical vitamins resulted in a 140% increase in epidermal thickness; an increase in gene and protein expression of collagen I, glycosaminoglycans (GAGs) and growth factors such as VEGF, EGF and FGF7. The collagen fibre bundles were increased, thickened, and more loosely woven in both the papillary and reticular dermis. CONCLUSION: We were able to show that PCI modulates gene expression in skin of those genes that are relevant for extracellular matrix remodelling.

[The role of erythropoietin in improvement of wound healing]. / Die Rolle von Erythropoietin bei der Verbesserung der Wundheilung.

Pleiotropic substances are characterized by their versatile and complex range of actions which makes them potential new active agents for the therapy of wounds. Besides its known effect to increase red blood cell production, the glycoprotein hormone erythropoietin (EPO) has been found to demonstrate a tissue protective effect in several other organs. The administration of EPO during skin wound healing is most likely essentially based on its cytopotective, proangiogenic, antiapoptotic and antiinflammatory effects. Herein EPO stimulates a coordinated interaction of different types of cells at a low or only a single dose. This review article aims to present the advantages and disadvantages of EPO administration in different experimental models to study the healing and regeneration processes of the skin and discusses possible clinical applications.

[Stem cells from fatty tissue : A new resource for regenerative medicine?]. / Stammzellen aus dem Fettgewebe : Eine neue Ressource für die regenerative Medizin?

While stem cells derived from the bone marrow are well-known in clinical medicine, fatty tissue as a source of mesenchymal stem cells is still the subject of recent research. However, adipose-derived stem cells (ASC) are not only harvested less invasively, i.e. via minimally invasive liposuction, but also yield higher numbers of multipotent stem cells.Due to cell-cell interactions and also because of the very favorable secretion profile of growth factors and cytokines ASCs displayed an extraordinary regenerative potential in recent preclinical and clinical applications and achieved a significantly better healing in ischemic muscle, heart, and brain insults and in impaired wound healing. ASCs enhanced regeneration in skeletal tissues such as cartilage or bone. They also revealed immunomodulatory effects and improved the clinical status in immunological diseases.In conclusion ASCs are comparable to bone marrow-derived stem cells concerning possible applications in clinical medicine.

[Current perspective and limitations of autologous fat transplantation--"consensus meeting" of the German Society of Plastic, Reconstructive and Aesthetic Surgeons at Hannover; September 2009]. / Möglichkeiten und Grenzen der autologen Fetttransplantation--"Consensus Meeting" der DGPRAC in Hannover, September 2009.

One hundred years after the first description of autologous fat transplantation, this technique is receiving renewed attention. Initially, critically reviewed by plastic surgery societies, particularly those in the United States, the transfer of autologous fat was recently addressed at the September 2009 annual meeting of the German Society of Plastic Reconstructive and Aesthetic Surgeons in Hannover. In this consensus meeting, the panel reviewed both the current status of autologous fat transfer as well as established data concerning this evolving practice. In Germany, autologous fat transplantation is regulated by the Law on Tissue Transfer and Processing (Gewebegesetz). In an effort to facilitate future comparisons it is mandatory to describe harvesting, processing and reinjection techniques in detail. The consensus panel concluded that fat should be harvested using low vacuum settings and then transplanted in thin layers (Evidence V). Quantification of transplanted fat can best be performed by MRI (Evidence level III). Limited clinical studies are available with only some reaching a level of evidence II. At present, risk associated with autologous fat transplantation is considered to be minor. Tumor induction by autologous fat grafting is not proven. New techniques like stem cell enriched fat grafts may offer new promise for the Plastic and Reconstructive Surgeon.

Percutaneous collagen induction. Scarless skin rejuvenation: fact or fiction?

Photoageing is generally treated by ablative procedures that injure the epidermis and basement membrane, and lead to fibrosis of the dermis. Percutaneous collagen induction (PCI) therapy is an alternative treatment for photoaged skin that does not result in clinical signs of dermal fibrosis. In this study, the immediate effects of PCI on the skin were assessed, including the systemic inflammatory response and the production and gene expression of transforming growth factor (TGF) isoforms beta1, beta2 and beta3. Eighty rats were split into four groups: group 1 (n = 24; PCI plus skin care); group 2 (n = 24; skin care only); group 3 (n = 24; PCI only) and group 4 (n = 8; controls). Microarray analysis showed that TGF-beta3, an essential marker for preventing scarring, was upregulated and expressed for 2 weeks postoperatively. PCI might offer a regenerative therapy to improve skin appearance and quality and to improve or even prevent scarring.

A normothermic perfusion bioreactor to preserve viability of rat groin flaps extracorporally.

INTRODUCTION: Various attempts have been made to prolong tissue survival ex vivo. To achieve an adequate ex vivo condition for flap perfusion at normothermic temperatures in a bioreactor model, a suitable perfusion solution is necessary. The main purpose of our setting at 37 degrees C was to produce conditions under which multilineage stem cells from adipose tissue could differentiate. This study was designed to evaluate the effect of permanent perfusion on fat flaps of the rat. MATERIALS AND METHODS: We elevated an epigastric adipofascial flap based on the inferior superficial epigastric vessels bilaterally in male Lewis rats and connected it to a bioreactor. The system was run by a cable pump and filled either with Hannover or Eurocollin's solution with or without permanent perfusion for 10 days. The lactate dehydrogenase (LDH) level in each solution was analyzed every 48 hours, assuming that injured cells emit this enzyme to the extracellular space and consequently to the perfusion solution. Histological samples were analyzed at the end of each trial. RESULTS: There was a continuous significantly greater LDH level (P < .001) in bioreactors perfused with Hannover than with Eurocollin's solution. The nonperfused bioreactors showed a similar finding with lower levels compared with their perfused equivalents. Histological examination revealed significantly better preserved (P < .001) fat tissue structures in Hannover solution-perfused specimens. CONCLUSION: Because LDH has a half life of 24 hours, ongoing production of this enzyme for 10 days is a marker for an injured tissue consisting of viable cells. Bioreactors run with Hannover solution showed significantly higher LDH levels. Histological analyses revealed intact cells preserved in Hannover solution. Thus, Hannover solution seemed to be superior to Eurocollin's solution to keep flaps viable under normothermic conditions. The presented model facilitated fat tissue conservation under normothermic conditions and represented a foundation for further studies on the differentiation of vascularized fat tissue.

[Efficient production of transfected human keratinocytes under serum-free and feeder layer-free conditions]. / Effiziente Herstellung transfizierter humaner Keratinozyten unter serum- und Feederlayer-freien Bedingungen.

PURPOSE/BACKGROUND: Keratinocyte transplantation after burn injury and in chronic wound treatment is a potentially useful method in clinical practice. As transfer of keratinocytes is easily monitored and gene expression is controllable by topical administration of inductors, keratinocyte cultures are an especially interesting medium for gene therapeutic approaches far above of wound healing applications. A major obstacle is the standardization of keratinocyte preparation and maintenance of pure proliferative cultures for clinical application. The best outcomes in previous protocols were obtained using fibroblasts as a feeder layer, a requirement for long-term expanded cultures. Cell expansion and a high purity of keratinocytes are prerequisites for clinical transfer studies. Here, we describe a human keratinocytes preparation method that allows cell proliferation and expansion in culture without a feeder layer. MATERIALS AND METHODS: Human keratinocytes were prepared from skin biopsies and cultured on untreated plastic culture dishes using Waymouth medium the first days followed by a change to a commercially available serum-free keratinocyte medium. The cells were characterized morphologically followed by transfection. For positive selection, transfected cells were selected by the cotransfection system pMACS Kk and magnetic cell sorting. RESULTS: Transfection rates were determined by expression of GFP vector which were 35%. The usage of magnetic cell sorting resulted in positive selection of transfected cells. Positive cells were able to adhere and proliferate after the sorting procedure. High viability and expansion of plastic adherent keratinocytes was achieved allowing up to 5 passages without signs of senescence and the doubling times were 3-5 days. The cells displayed typical keratinocyte morphology and immunostaining confirmed high keratinocyte purity. The number of contaminating fibroblasts was low. CONCLUSION: Here, we describe an efficient and inexpensive method for a standardized human keratinocyte isolation without the need of a fibroblast feeder layer. This protocol may facilitate the clinical application of cell based therapies in burn injuries or chronic wounds using keratinocytes.

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration.

OBJECTIVE: In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20-mm sciatic nerve defect in rats. MATERIALS AND METHODS: We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. RESULTS: Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S-100 and neurofilament indicated existence of Schwann cells and axonal re-growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell-free constructs based on vein and spider silk was highly effective in nerve regeneration. CONCLUSION: We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re-growth in a long-distance model for peripheral nerve regeneration.

[Innovative wound therapy and skin substitutes for burns]. / Innovative Wundtherapie und Hautersatz bei Verbrennungen.

The success of modern burn therapy is based mainly on special burn intensive care, topical treatment, early eschar excision, and wound closure by immediate skin grafting or skin substitutes. This paper describes the current state of wound care and skin substitutes in burn therapy.

[Apoptosis and its role in plastic surgery]. / Die Apoptose und ihre Bedeutung in der Plastischen Chirurgie.

Apoptosis and cell proliferation are the main mechanisms of cell homeostasis. The pathogenesis of approximately 70 % of all diseases results from an imbalance between these two counterparts. Therefore, research on apoptosis is a main target in biological and clinical fields. Many signalling pathways and proteins involved in their regulation have been characterized. In order to evaluate the relevance of apoptosis in plastic surgery, the literature was reviewed for its impact on ischemia and reperfusion concerning flap surgery as well as wound healing and angiogenesis. Furthermore, the relevance of apoptosis in ageing, allotransplantation and tumors was investigated. In all subsections of plastic surgery, a high impact was identified. More studies on the influence and regulation of apoptosis can bring further understanding on the disease patterns of plastic surgery and other specialties as well as the development of new therapeutic options. Research focusing on apoptosis is therefore an essential means for the advancement of and future trends in plastic surgery.

Identification of an unconventional nuclear localization signal in human ribosomal protein S2.

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.

[In vitro fabrication of tendon substitutes using human mesenchymal stem cells and a collagen type I gel]. / Herstellung von Sehnenkonstrukten aus humanen mesenchymalen Stammzellen und einem Kollagen Typ I Gel.

BACKGROUND AND PURPOSE: Reconstruction of tendon tissue is problematic in many cases. Since direct tendon suture is often impossible, major reconstruction with the use of free tendon transplants or tendon transposition is necessary. Important motor units often have to be sacrificed for reconstructive purposes. In this study we investigated whether long tendon-like substitutes can be fabricated in vitro from human mesenchymal stem cells (MSCs) and a collagen type I gel when cultured under cyclic stretching conditions. MATERIAL AND METHODS: MSCs were obtained from bone marrow aspirates of the iliac crest. Cells were suspended in a collagen type I gel and polymerized in a glass-cylinder with defined size. The fabricated tendon substitutes underwent static stretching for 14 days followed by cyclic stretching for 21 days in a special manufactured bioreactor. Non-stretched substitutes served as a control. RESULTS: Macroscopically the stretched tendon substitutes showed an increased opacity and a smoother surface structure compared to the non-stretched control. The stretched substitutes displayed more spindle-shaped, longitudinal orientated cells, a tendon-like organization of the collagen matrix, and a parallel organization of the collagen fibers when stained with Hematoxylin/Eosin and Elastica. CONCLUSION: Long tendon substitutes could be fabricated from MSCs and a collagen type I gel by cyclic stretching and showed tendon-like parallel collagen fibers and spindle-shaped cells. The use of MSCs in combination with adequate scaffold materials has great therapeutic potential for the development of autologous transplantable tendon substitutes.

[Influence of foam- and tubing material of the vacuum assisted closure device (v.a.C.) on the concentration of transforming growth factor Beta 1 in wound fluid]. / Einfluss des Schwamm- und Schlauchmaterials der Vacuum Assisted Closure Device (V.A.C.) auf die Konzentration von Transforming Growth Factor beta 1 in Wundflüssigkeit.

BACKGROUND AND PURPOSE: The Vacuum Assisted Closure device (V.A.C.) is commonly used for the treatment of problematic wounds. Furthermore, wound fluid can be easily collected with this device for research purposes. However, there is inadequate information as to whether the measurement of biomoieties of importance to wound healing is affected by the exposure of wound fluid to V.A.C. components, namely Polyurethane-foam and tubing. This study is an attempt to evaluate whether exposure of wound fluid to either V.A.C.-components affects concentrations of transforming growth factor beta 1 (TGF-b1) in wound fluid. MATERIAL AND METHOD: Wound fluid was gathered from five decubital ulcer patients using the foil-technique and was exposed to sterile pieces of the V.A.C. Polyurethane-foam, tubing material or nothing for zero, one or five hours. Saline served as control. The concentration of TGF-b1 was measured using sandwich-ELISA. The resulting data were analyzed using two-way ANOVA, Newman-Keuls and Bonferroni t-Test. RESULTS: The concentration of TGF-b1 decreased significant in all three groups during the five hours of the experiment (p < 0.05). There was no significant decrease in TGF-b1 concentration at any time point in-between the groups. CONCLUSION: From this study, we conclude that wound-fluid collected from the V.A.C.-device via the polyurethane-foam or tubing for purposes of analyzing concentrations of TGF-beta 1 should not be different from fluid collected using the foil technique.

Structure and function of bone marrow hemopoiesis: mechanisms of response to ionizing radiation exposure.

It is the purpose of this presentation to review the unique structure and function of bone marrow anchored hematopoiesis in their significance for its response mechanisms to an exposure to ionizing radiation. The ultimate objective of bone marrow hematopoiesis is to maintain in the peripheral blood a constant level of the different blood cell types (erythrocytes, granulocytes, platelets, lymphocytes, etc.). All of them have their particular turnover kinetics (such as granulocytes 120 x 10(9)/d, erythrocytes 200 x 10(9)/d or thrombocytes 150 x 10(9)/d), are semi-autonomous in their steady state regulatory mechanisms and dependent on a life-long supply of mature cells from a stem cell pool with unlimited replicative and pluripotent differentiative potential. The present knowledge of hematopoietic cellular renewal is the result of years of basic experimental and clinical studies using radionuclides in various metabolic forms including (59)Fe, (32)P (DF (32)P), (51)Cr, (131)I, (60)Co, (3)H ((3)HTdR) and (14)C ((14)CTdR). To understand the physiology but in particular the radiation-pathophysiology, it is essential to recognize in detail the infrastructure of the bone marrow as a distinct unit. Indispensable for a life-long cell production is the capsule of the marrow - the bone cortex -, the arterial supply of blood connected to the sinusoidal microvascular architecture with its sinusoids contorti and recti as well as the central (cell collecting) sinusoids. It is further of importance to recognize the significance of nerval regulation of blood flow, characterized by myelinated and unmyelinated nerve fibers. The type of unique lining cells of the sinusoids is the prerequisite for the cell traffic between the hemopoietic parenchyma and the blood. This in turn cannot be achieved without an alternative opening and closing of the sinusoidal segments which - in turn - requires a rigid long capsule to assure an - in toto - constant volume of each bone marrow unit. If a bone marrow unit is exposed to ionizing radiation, a perturbance of the balance between cellular growth pressure and blood flow dynamics can be observed, resulting in a special type of bone marrow hemorrhage and an "excess cell loss" that may result in an non-thrombopenic exhaustion of the stem cell pool. Of great importance is the question as to the mechanisms that allow the bone marrow hemopoiesis to act as one cell renewal system although the bone marrow units are distributed throughout more than 100 bone marrow areas or units in the skeleton. The observation that "the bone marrow" acts and reacts as "one organ" is due to the regulatory mechanisms: the humeral factors (such as erythropoietins, granulopoietins, thrombopoietins etc.), the nerval factors (central nervous regulation) and cellular factors (continuous migration of stem cells through the blood to assure a sufficient stem cell pool size in each bone marrow "sub-unit"). It should be recalled that the bone marrow functions as a physiological chimera and becomes established by the hematogeneic seeding of stem cells to a mesenchymal matrix during embryogenesis. The repopulation of the bone marrow after partial body irradiation, after strongly inhomogeneous radiation exposure or after total body exposure with stem cell transplantation can well be considered as a repetition of the embryogenesis of bone marrow hemopoiesis with the key element of stem cells migrating via the blood to stromal sites of the marrow prepared to accept stem cells to home and start their replication and differentiation if the micro-environmental quality permits. In summary, the radiation biology of bone marrow hemopoiesis requires a thorough understanding of the physiology and pathophysiology of structure, function and regulation not only of the process of cellular renewal but also of the intricate infrastructure.

NoBP, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth.

Secreted and nuclear forms of fibroblast growth factor 3 (FGF3) have opposing effects on cells. The secreted form stimulates cell growth and transformation, while the nuclear form inhibits DNA synthesis and cell proliferation. By using the yeast two-hybrid system we have identified a nucleolar FGF3 binding protein (NoBP) which coimmunoprecipitated and colocalized with FGF3 in transfected COS-1 cells. Characterization of the NoBP binding domain of FGF3 exactly matched the sequence requirements of FGF3 for its translocation into the nucleoli, suggesting that NoBP might be the nucleolar binding partner of FGF3 essential for its nucleolus localization. Carboxyl-terminal domains of NoBP contain linear nuclear and nucleolar targeting motifs which are capable of directing a heterologous protein beta-galactosidase to the nucleus and the nucleoli. While NoBP expression was detected in all analyzed proliferating established cell lines, NoBP transcription was rapidly downregulated in the promyelocytic leukemia cell line HL60 when induced to differentiate. Analysis on the expression pattern of NoBP mRNA throughout the cell cycle in HeLa cells synchronized by lovastatin demonstrated a substantial upregulation during the late G(1)/early S phase. NoBP overexpression conferred a proliferating effect onto NIH 3T3 cells and can counteract the inhibitory effect of nuclear FGF3, suggesting a role of NoBP in controlling proliferation in cells. We propose that NoBP is the functional target of nuclear FGF3 action.