Transcriptional control of p53 expression participates in the generation of appropriate levels of active p53 in response to mitogenic stimulation. This prompted us to study the role of a putative AP-1 and a NF-kB motif in the human p53 promoter for transcriptional regulation. We show that mutation of the AP-1 or the NF-kB motif abolishes transcription from the human p53 promoter in HeLa, HepG2 and adenovirus type 5 E1-transformed 293 cells. In comparison, mutation of the previously characterized Myc/Max/USF binding site in the human p53 promoter reduces the transcription rate ®vefold. The AP-1 motif in the human p53 promoter binds c-Fos and c-Jun and the NF-kB motif binds p50 NF-kB1 and p65 RelA . The cooperative nature of transcriptional activation by these factors was documented by repression of c-fos or NF-kB1 translation: Pretreatment of the cells with a cfos or p50 NF-kB1 antisense oligonucleotide suppresses transcription from the human p53 promoter completely. In addition, we show that (a) the level of endogenous p53 mRNA and (b) transcription from the strictly p53-dependent human mdm2 promoter are reduced in the presence of c-fos, c-jun, p50 NF-kB1 , p65 RelA or c-myc antisense oligonucleotides, underscoring the importance of these transcription factors for the expression of functional p53.

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.

Bone morphogenetic proteins (BMPs) play a decisive role in bone development and osteogenesis. In the past they have been the subject of widespread research and clinical trials as stimulants of bone growth. Although recently recombinant human BMP-2 (rhBMP-2) has been chemically immobilized on implant surfaces leading to enhanced bone growth and accelerated integration in vivo, the non-covalent immobilization of proteins on metal surfaces is still poorly understood, since the oxide layers on metals like titanium, stainless steel or cobalt chromium alloys are poor adsorbents of proteins. Protein binding surfaces could either be generated by linking ionic groups (ion-exchange surface) or by coupling hydrophobic residues (hydrophobic interacting surface, HIS) to the surface. In this paper the preparation of protein adsorbing surfaces on titanium and cobalt chromium molybdenum alloy for the adsorption of rhBMP-2 and ubiquitin will be described. rhBMP-2 and ubiquitin are bound extremely tight to surfaces containing propyl or hexyl groups of a certain surface concentration and are slowly released over a range of at least 24 ± 100 days making such surfaces applicable as long-term drug delivery devices for enhancing bone growth or implant integration.Knochenmorphogenetische Proteine (BMPs) spielen eine entscheidende Rolle in der Entwicklung der Organismen und der Osteogenese. In der Vergangenheit waren sie in der Stimulierung des Knochenwachstums Thema zahlreicher Untersuchungen und klinischer Studien. Obwohl ku Èrzlich rekombinantes humanes BMP-2 (rhBMP-2), das auf Implantatoberfla Èchen chemisch immobilisiert wurde zu versta Èrktem Knochenwachstum und zur beschleunigten Integration in vivo fu Èhrten, ist die nicht-kovalante Immobilisierung von Proteinen auf metallischen Oberfla Èchen bisher nur wenig verstanden, da die Oxidschichten auf Metallen wie Titan, rostfreiem Stahl oder Kobalt-Chrom-Legierungen Proteine nur schlecht adsorbieren. Proteinbindende Oberfla Èchen ko Ènnen entweder durch die Kopplung von geladenen Moleku Èlen (Ionenaustauscheroberfla Èche) oder von hydrophoben Resten (hydrophob interagierende Oberfla Èchen, HIS) an die Oberfla Èche hergestellt werden. In dieser Arbeit wird die Herstellung von Protein adsorbierenden Oberfla Èchen auf Titan und einer Kobalt-Chrom-Molybda Èn Legierung fu Èr die Adsorption von rhBMP-2 und Ubiquitin beschrieben. RhBMP-2 und Ubiquitin werden auûerordentlich stark an Oberfla Èchen gebunden, die Propyl-oder Hexylgruppen bestimmter Oberfla Èchenkonzentration tragen und werden langsam u Èber einen Zeitraum von 24 ± 100 Tagen von den Oberfla Èchen wieder freigesetzt, so daû sich diese als langzeit ¹drug delivery devicesª zur Anregung des Knochenwachstums und zur Beschleunigung der Implantatintegration eignen.

The binding of rhBMP‐2 to its receptors, the signal transduction cascade and the final responses of bone cells, osteoprogenitor cells and derived cell lines is of high fundamental and clinical interest. In this report concentration‐response curves of the osteoblast cell line MC3T3‐E1 under influence of rhBMP‐2 was investigated. The biological response of the cells (corresponding to a down‐stream effect of the receptor state‐function) was monitored in pilot experiments by the MC3T3‐cell alkaline phosphatase‐induction test (MC3T3‐cell ALP‐induction test). It is shown that the MC3T3‐cell ALP‐induction test is a good tool for measuring biologically active recombinant human BMP‐2 (rhBMP‐2) in crude extracts of E. coli as well as in highly purified form. In addition this test is very sensitive to chemically induced structural changes of rhBMP‐2 such as those resulting from a radiolabeling of rhBMP‐2 by the Bolton‐Hunter procedure. The latter procedure reduces the biological activity of rhBMP‐2 by a factor of 3–4. The measured concentration‐response curves could all be non‐linearly fitted to a rectangular hyperbola. The half‐maximal saturation, K0.5, is found between 30–100 nM rhBMP‐2 (= 0.8–2.5 μg/ml). The effect of rhBMP‐2 shows a plateau i.e. maximal response at ca. 300–1000 nM rhBMP‐2 (= 8–25 μg/ml). The data thus indicate a non‐cooperative binding‐response behavior. This was unexpected since BMP‐2 binds simultaneously to two cooperating receptors of type 1 and type 2. However in the very low concentration range of employed rhBMP‐2 a variable response of the cells was measured so that a full exclusion of cooperativity cannot be concluded at the present time. This will be clarified by future experiments.

Previously it has been shown that recombinant human bone morphogenetic protein (rhBMP‐2) can be chemically immobilized by “anchor molecules” on titanium surfaces for serving as a drug delivery device. This opened the question of whether the insoluble immobilized rhBMP‐2 retained its activity in comparison to the same amount of soluble rhBMP‐2 included with the implant samples. Electropolished titanium miniplates (10 × 6 × 0.8 mm) were “surface‐enhanced” by a novel treatment with chromosulfuric acid and then coated with a total amount of 150–200 ng rhBMP‐2 prepared by recombinant technology. Periosteal flaps (7 × 20 mm) were detached and isolated from the anterior surface of the tibiae of adult rabbits and wrapped around the titanium sample plates which were then implanted in the M. gastrocnemius. In the first experimental group various controls without rhBMP‐2 were combined (n = 12). In the second experimental group implants with chemically immobilized rhBMP‐2 (n = 8) were compared with implants to which non‐immobilized soluble rhBMP‐2 was added (n = 8). Animals were sacrificed after 28 days and a quantitative evaluation was carried out by means of serial sections. Untreated control plates showed bone formation in 2/12 implants, rhBMP‐2 coated implants in 6/8 and implants with free rhBMP‐2 administered subperiostally in 8/8 cases. In the case of rhBMP‐2 coated implants the induced bone had direct contact to the implant in all cases while in the group with free administered rhBMP‐2 the bone had no contact to the implant in two cases, but was separated by a fibrous capsule. Bone volume, bone surface area, and trabecular number displayed no difference between the two rhBMP‐2‐groups. However, in the biocoated group a tendency to an increase in the bone‐implant contact area was evident. No differences in osteoid area, osteoid perimeter and eroded perimeter were detected. We conclude that in the case of non‐immobilized rhBMP‐2 there is the danger for formation of fibrous tissue between the implant and the newly formed bone and in addition the generation of ectopic bone at inappropriate places. In contrast chemically immobilized rhBMP‐2 does not have these drawbacks and at the same time displays a biological activity on surfaces similar to that of soluble rhBMP‐2 demonstrating that biomaterial surfaces can be tailored for a selective and specific interaction with the target tissue.