Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknüllt protein.

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene.

We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.

Murine DNA polymerase beta gene: mapping of transcription initiation sites and the nucleotide sequence of the putative promoter region.

The nucleotide sequence of the region (total, 2,512 base pairs [bp]) from intron 2 to the 5'-flanking region was determined for the mouse DNA polymerase beta genomic clone, and the 300-bp region from intron 1 to the 5'-flanking region was also sequenced for the rat clone. At 51 bp upstream from the ATG codon which was previously suggested to be the translation initiation codon for the rat cDNA sequence, we found another ATG in the same reading frame in both mouse and rat genes. Three major transcription initiation sites (cap sites) each for rat and mouse DNA polymerase beta mRNAs were localized precisely by primer extension analysis at 51, 41, and 0 bp upstream from the first ATG codon, suggesting that this codon is used for translation initiation. The 400-bp region around exon 1 was extremely G + C rich (about 70%). Although neither a TATA box nor a CAAT box was found within the 500-bp region upstream of the 5'-most cap site, triple repeats of 5'-CCGCCC were found within the 100-bp region flanking the cap site.

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5'-flanking region.

The genomic and cDNA clones for a Drosophila melanogaster proliferating cell nuclear antigen (PCNA) (cyclin) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide was interrupted by a single short intron of 60 base pairs (bp), and about 70% of the deduced amino acid sequence of the Drosophila PCNA was identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome. The PCNA mRNA was detected at a high level in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The major transcription initiation site (cap site) was localized at 89 bp upstream from the ATG codon. Neither a TATA box nor a CAAT box was found within the 600-bp region upstream of the cap site. Clusters of 10 bp of sequence similar to the binding sites for Drosophila proteins containing homeodomains were found in the region from -127 to -413. DNase I footprint analysis revealed that the Drosophila homeodomain proteins coded by even-skipped and zerknüllt genes can specifically bind to these sites. These results suggest that the expression of the PCNA gene is under the control of genes coding for homeodomain proteins.

E2F-dependent transcription of the raf proto-oncogene during Drosophila development.

D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5'-flanking region of D-raf, four sequences (E2F sites 1-4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf-lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.

Ectopic expression of human p53 inhibits entry into S phase and induces apoptosis in the Drosophila eye imaginal disc.

Transgenic flies in which ectopic expression of human p53 was targeted to the Drosophila eye imaginal disc were established. On sectioning of adult fly eyes which displayed a severe rough eye phenotype, most ommatidia were found to be fused and irregular shapes of rabdomeres were observed. In addition, many pigment cells were lost. In the developing eye imaginal disc, photoreceptor cell differentiation was initiated normally despite the ectopic expression of p53. However, expression of p53 inhibited cell cycle progression in eye imaginal disc cells and the S phase zone (the second mitotic wave) behind the morphogenetic furrow was almost completely abolished. Furthermore, expression of p53 induced extensive apoptosis of eye imaginal disc cells, and co-expression of baculovirus P35 in the eye imaginal disc suppressed the p53-induced rough eye phenotype. These results are consistent with the known functions of human p53 and indicate the existence of signaling systems with elements corresponding to human p53 in Drosophila eye imaginal disc cells. Genetic crosses of transgenic flies expressing p53 to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the p53-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify novel targets of p53 and its positive or negative regulators in Drosophila.

Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration.

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

In vivo evaluation of ocular inflammatory responses in experimental diabetes.

AIMS: Diabetic patients may have abnormal inflammatory reactions to foreign or endogenous stimuli. This study was designed to evaluate inflammatory reactions in the diabetic eye through retinal leucocyte dynamics in the inflamed eyes of diabetic rats. METHODS: Three weeks after diabetes induction in Long-Evans rats, endotoxin induced uveitis was produced by footpad injection of lipopolysaccharide (LPS). After LPS injection, leucocyte behaviour was evaluated in vivo by acridine orange digital fluorography. RESULTS: The number of rolling leucocytes increased in a biphasic manner at 12 hours and 48 hours. The number of leucocytes accumulating in the retina reached a peak at 72 hours. The maximal numbers of rolling and accumulating leucocytes in the diabetic retina decreased by 56.3% (p<0.01) and 46.7% (p<0.0001), respectively, compared with the non-diabetic retina. The levels of mRNA expression of adhesion molecules in the retina, which were upregulated after LPS injection, were also lower in diabetic rats than in non-diabetic rats. CONCLUSION: This study is the first to show that endotoxin induced inflammation is disturbed in the diabetic eye, based on evidence that the leucocyte-endothelial cell interactions stimulated by LPS were suppressed in the diabetic retina. These findings support the theory that ocular inflammatory reactions are impaired in diabetic patients.

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.

The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes.

We have confirmed that the DNA replication-related element (DRE) consisting of an 8-bp palindrome, TATCGATA, and not neighboring sequences, are responsible for activating promoters of the Drosophila melanogaster (Dm) PCNA (proliferating cell nuclear antigen)- and DNA polymerase alpha-encoding genes in both cultured cell and transgenic fly systems. We have so far found 153 copies of DRE in the Dm gene database. 73 of them are concentrated within the 600-bp upstream regions from the transcription start points of 61 genes. Interestingly, many of these genes are involved in either DNA replication, transcription, translation, signal transduction, cell cycle or other putative regulatory functions, and are possibly related to cell proliferation. It seems likely that DRE is an element common to the regulation of cell-proliferation-related genes, although their expression patterns may be different depending on which of regulatory elements other than the DRE are combined.

Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF).

The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, we have obtained a clone encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). We termed the protein Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro could be confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, we performed immunofluorescent staining of Drosophila Kc cells and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila.

cDNA cloning and expression during development of Drosophila melanogaster MCM3, MCM6 and MCM7.

cDNAs encoding three Drosophila melanogaster MCM proteins, DmMCM3, DmMCM6 and DmMCM7, candidates of DNA replication-licensing factors, were cloned and sequenced. The deduced amino-acid sequences displayed 60, 59 and 68% identities with the respective Xenopus laevis homologues, XMCM3, XMCM6 and XMCM7. Six members of the D. melanogaster MCM family were found to share 31-36% identities in their amino-acid sequences, and to possess the five common domains carrying conserved amino-acid sequences as reported with X. laevis MCM proteins. DmMCM3, DmMCM6 and DmMCM7 genes were mapped to the 4F region on the X chromosome, the 6B region on the X chromosome and the 66E region on the third chromosome, respectively, by in situ hybridization. Contents of their mRNAs were proved to be high in unfertilized eggs and early embryos (0-4h after fertilization), then decrease gradually by the 12h time point, with only low levels detected at later stages of development except in adult females. This fluctuation pattern is similar to those of genes for proteins involved in DNA replication, such as DNA polymerase alpha and proliferating cell nuclear antigen, suggesting that expression of DmMCM genes is under the regulatory mechanism which regulates expression of other genes involved in DNA replication.

Molecular cloning and expression during development of the Drosophila gene for the catalytic subunit of DNA polymerase epsilon.

We have cloned the genomic DNA and cDNA of Drosophila DNA polymerase epsilon (pol-epsilon) catalytic subunit (GenBank No. AB035512). The gene is separated into four exons by three short introns, and the open reading frame consists of 6660 base pairs (bp) capable of encoding a polypeptide of 2220 amino acid residues. The calculated molecular mass is 255018, similar to that of mammalian and yeast homologues. The deduced amino acid sequence of the pol-epsilon catalytic subunit shares approximately 41% identity with human and mouse homologues as well as significant homology those of C. elegans, S. cerevisiae and S. pombe. Similar to the pol-epsilon catalytic subunits from other species, the pol-epsilon catalytic subunit contains domains for DNA polymerization and 3'-5' exonuclease in the N-terminal region, and two potential zinc-finger domains in the C-terminal regions. Interestingly, a 38 amino acid sequence in the C-terminal region from amino acid positions 1823 to 1861 is similar to the site for Mycoplasma ATP binding and/or ATPase domain (GenBank No. P47365). Northern hybridization analysis indicated that the gene is expressed at the highest levels in unfertilized eggs, followed by zero to 4h embryos and adult females, and then embryos at other embryonic stages, instar larva stages and adult males. Low levels of the mRNA were also detected at the pupa stage. This pattern of expression is similar to those of DNA replication-related enzymes such as DNA polymerase alpha and delta except for the high level of expression in adult males.

Ectopic production of lipotropin by cancer.

Lipotropin (LPH) has been evaluated as a potential tumor marker using a sensitive beta melanocyte-stimulating hormone (beta MSH) radioimmunoassay. All 79 acetic acid extracts of carcinomas of lung, colon, stomach, esophagus and breast contained LPH in concentrations greater than blood; 61 of 79 extracts contained LPH in larger amounts than control tissues from patients without cancer. In a blind prospective study, plasma LPH was quantified in 107 patients admitted for work-up because of an abnormality on a chest roentgenogram. Thirty-one of 33 patients subsequently diagnosed as having benign lesions had plasma LPH within the 95 per cent confidence limits of normal subjects whereas 28 (36 per cent) of the 74 patients subsequently diagnosed histologically as having primary lung carcinoma had elevated levels. In control studies, 13 of 100 patients with chronic obstructive pulmonary disease had elevated plasma LPH levels; three of the 13 with elevated levels and four with normal levels have been diagnosed, during the two years of follow-up, as having lung carcinoma. In control studies of 23 patients with granulomatous lung disease, 22 had normal levels of LPH. In those with carcinoma of the colon elevated plasma LPH levels were observed in two of 21 untreated patients and in 11 of 61 patients receiving noncurative chemotherapy. Elevated plasma LPH levels were also observed in 10 of 59 patients with breast cancer, eight of 28 with pancreatic cancer, eight of 22 with gastric or esophageal cancer, six of 16 with renal cancer, four of eight with prostatic cancer, one of seven with cervical cancer and one of six with ovarian cancer. We conclude, an elevated LPH level is frequently observed in blood and tumor tissue from patients with various types of carcinoma.

Morphologic evidence of accelerated closure of the ductus arteriosus in preterm infants.

In preterm infants, closure of the ductus arteriosus (DA) is often delayed, especially in those with respiratory distress syndrome (RDS). However, it has been suggested that functional closure of the DA may occur as early as 24 hours of age in some preterm infants exposed to intrauterine stress, and this is usually associated with decreased incidence of RDS. This suggests that accelerated maturation of the DA as well as of the lungs occurs in utero. Accordingly, histologic evidence of accelerated maturation of the DA was sought in a prospective autopsy study of 55 preterm infants ranging in gestational age from 19 to 32 weeks. There were four infants with clinically closed DA which showed histologic evidence of closure. The birth weight of these four infants ranged from 750--1,100 gm, the gestational age ranged from 24--32 weeks, and age of death was 39 hours to 6 days. The immediate causes of death were intracerebral hemorrhage or intrapulmonary hemorrhage, or both. Obstetric complications included chronic second trimester vaginal bleeding, abruptio placenta, malnutrition, diabetes, pulmonic stenosis of moderate degree, and chronic hypertension. These findings support the hypothesis that in some preterm infants exposed to chronic intrauterine stress, maturation of the DA is accelerated. This may result clinically in effective postnatal closure of the DA.

Herpes simplex viral infection in human neonates: an immunohistochemical and electron microscopic study.

Specimens obtained at autopsy from six neonates with herpes simplex virus (HSV) infections were examined microscopically, electron microscopically, and immunohistochemically. Coagulative necrosis with inclusions was found in the livers and adrenal glands in all cases, as well as in various other organs, including the spleen, bone marrow, lungs, esophagus, tongue, and thymus, in some cases. Distinct hemorrhagic diathesis was found in three cases. No characteristic clinical findings, such as skin rashes or elevated titers of the antibody to HSV, were found, and clinical diagnosis was therefore difficult. In three cases isolation and typing of the causative virus were performed virologically, and type 1 HSV (HSV-1) was identified as the causative virus. Immunohistochemically, the type and distribution of the virus were evaluated in all cases with type-specific antisera to types 1 and 2 (HSV-2) antigens by the peroxidase-antiperoxidase method. In five cases the infections were found to be due to HSV-1 and in only one case to HSV-2. In the placenta in one case of HSV-2 infection, HSV antigen was demonstrated in the chorionic villi. Electron microscopic study confirmed the existence of viral particles in the placenta in that case and, thus, the possibility of a transplacental route of infection.

Neovascularity of a tracheal prosthesis/tissue complex.

Permanent bioincorporation of a microporous tracheal prosthesis will require a stable blood supply to connective tissue supporting an epithelial surface. In experience with over 80 tracheal implants in dogs, we have observed that end-on ingrowth and epithelialization does not occur in the absence of lateral ingrowth, epithelialization is marked by the appearance of a subepithelial network of vessels, and this process must be well advanced by 6 to 8 weeks for long-term stability. These observations were extended by using microangiography to delineate the blood supply of the prosthesis/tissue complex. Six implants of bioelectric polyurethane with 10% gentamicin (3 cm length, 2 cm diameter, 1 to 1.25 mm wall thickness, 60 to 120 mu micropore diameter) were interposed in the dog thoracic trachea and wrapped with an omental pedicle. The aorta was perfused with a barium suspension at elective sacrifice between 10 weeks and 21 months. Radiographs of specimens were correlated with bronchoscopic, gross, and histopathological findings. Neovascularity of the prosthesis/tissue complex can be described in three categories: outer capsule, prosthetic wall, and inner lining. Outer capsule vessels were oriented circumferentially immediately adjacent to the prosthetic wall. They resembled arteries up to 75 mu diameter on microscopy and appeared to develop from the omentum with connections developing to the bronchial circulation. Prosthetic wall vessels up to 75 mu with thin muscular walls were noted to traverse the porous prosthetic wall. The inner lining had a network of subepithelial vessels that connected to the lamina propria vasculature of the native trachea across the anastomoses with vessels up to 120 mu in diameter. We conclude that the omentum provides an immediate blood supply and a base for early connective tissue ingrowth. Epithelialization occurs as early as 3 weeks on the favorable bed, accompanied by vascular connections to the existing lamina propria tracheal vessels. This dual organization of blood supply with connections across the prosthetic wall is probably important to long-term stability of healing.

Histopathologic observations after short-term implantation of two porous elastomers in dogs.

This report describes the effects of pore size and material on soft tissue ingrowth of two medical-grade elastomers. Using the replamineform process, silicone rubber (SR) and bioelectric polyurethane (BEP) were rendered microporous with essentially the same microstructural pore configuration. Implants were prepared in each material having five pore size ranges: 18-25 microns, 30-45 microns, 75-95 microns, 60-120 microns, and 120-180 microns. Implants 1 cm X 1 cm X 1 mm were harvested at 1, 2, 4 and 12 weeks following subcutaneous implantation in mongrel dogs. Ingrowth of the 18-25 microns and 30-45 microns implants in both polymers consisted of histiocytes and dispersed fibrocytic proliferation during the first two weeks. By 12 weeks, the fibrocytic component had increased, but histiocytes remained the principal component of ingrown tissue. In contrast, initial ingrowth of the 75-95 microns, 60-120 microns and 120-180 microns implants showed increased fibrocytic proliferation and minimal histiocytic reaction. By 12 weeks, ingrowth into the larger-pore implants had progressed to broad bands of well organized collagenous stroma. Differences in the rate of tissue ingrowth were found to be related to both material and pore size. Less than 15% of the void spaces were infiltrated by 4 weeks in 18-25 microns and 30-45 microns SR implants, although this increased to approximately 50% by 12 weeks. In contrast, the 3 larger-pore SR implants and all pore sizes in the BEP implants were almost completely ingrown by 4 weeks.