Matti Vauhkonen

Distraction bone healing.

Bone formation by distraction was studied using three different experimental models: (1) Physeal distraction of the sheep radius was performed in 20 animals. (2) Distraction after osteotomy of the radius was carried out in 39 sheep. (3) Mandibular distraction after osteotomy was performed in 17 sheep. Formation of the organic matrix and osteogenesis were studied by radiographic, histologic, and biochemical methods as well as by electron microscopy. The mode of osteogenesis was essentially similar in all of these distraction models. Bone formation was preceded by organization of the collagenous matrix in the distraction area. In the beginning of the distraction, the gap was composed of a heterogeneous cell population, with large polymorphic fibroblast-like cells. The cells in the central part differentiated into fibroblasts, which remained functionally active as long as distraction proceeded. During physeal distraction, bone formed from the epiphyseal and metaphyseal sides as well as from the surrounding perichondrium. Also, in osteotomy distraction of both tubular bone and mandible, bone formed centripetally from the osteotomized bone ends toward the center of the gap. The organic matrix was composed almost solely of Type I collagen in the earliest stages, suggesting that the mode of osteogenesis differs from bone repair by fracture callus. The structure of the distracted segment was mainly lamellar trabecular. Corticalization of the lengthened bone segment occurred gradually after several months.

Collagen synthesis and mineralization in the early phase of distraction bone healing.

Corticotomy of the distal radius followed by gradual distraction by external fixation was performed on three sheep. Collagen synthesis and mineral deposition were analysed from sequential biopsies obtained from the center of the distraction area during the first 4 weeks of distraction. The whole distraction area was rapidly filled with organic matrix the amount of which, due to fluctuation in its nonprotein component, initially decreased from 88 to 66% of the level in control bone but gained its initial level in 4 weeks. Total protein in the matrix represented 70% of that in the control bone during the 4-week follow up period while the proportion of collagen of the total protein increased from 53 to 88%, a level comparable with the unoperated bone. Determination of the type of fibrillar collagen by characterization of their cyanogen bromide peptides showed that in the distraction area production of type II collagen does not occur but the heteropolymer type I (alpha 1(I)2 alpha 2(I)1) collagen represents almost totally the collagen synthesized. Deposition of mineral into the distraction gap was detectable already after 2 weeks and increased rapidly after 3 weeks of distraction. The results suggest that unlike in other processes, e.g., direct osteonal and callus-type bone repair, in distraction bone healing gradual distraction of osteotomized bone leads directly to synthesis of mature fibrous organic matrix of bone followed by its rapid mineralization.

Substrate Specificity and Activation Mechanisms of Collagenase from Human Rheumatoid Synovium

Substrate specificity studies of collagenase extracted from human rheumatoid synovium suggest that synovial pannus tissue overlying articular cartilage may not be particularly active in degradation of cartilage type II collagen, which, considering the poor inherent healing capacity of the articular hyaline cartilage, may exert a protective function against inadvertant tissue damage. Rheumatoid synovial tissue was also used to establish synovial fibroblast cell lines. Treatment of these cells in monolayer cultures with IL-1 leads to collagenase gene activation, increased collagenase production and an almost complete autoactivation of secreted collagenase. Interleukin-1 also activated stromelysin gene suggesting this as a possible mechanism effecting autoactivation. Latent human fibroblast and macrophage collagenase purified from culture medium were efficiently activated by phenylmercuric chloride but also by gold thioglucose, gold sodium thiomalate and HCIO. These new observations support the Cys73 switch activation mechanism. In contrast to neutrophil collagenase, the activation by gold(I) compounds and HCIO was associated with a change in the apparent molecular weight of the fibroblast procollagenase. In addition, gold(I) compounds rendered collagenase more susceptible to thermal denaturation. Thus the fibroblast-type interstitial collagenase, probably derived from fibroblast- and macrophage-like synoviocytes, seems to provide the predominant collagenolytic potential in human rheumatoid synovial tissue. Furthermore, the conditions in synovitis tissue may be such as to favor at least initial activation of collagenase synthesized and secreted in situ.

Collagenase Reserves in Polymorphonuclear Neutrophil Leukocytes from Synovial Fluid and Peripheral Blood of Patients with Rheumatoid Arthritis

Degradation of cartilage in rheumatoid arthritis (RA) may be in part due to release of collagenase from specific granules of polymorphonuclear neutrophil leukocytes (PMNs) during degranulation. We decided to study, not synovial fluid (SF) collagenase, but PMN collagenase reserves. PMN were isolated from parallel SF and peripheral blood (PB) samples obtained from 7-arthritis patients. PMNs were stimulated in vitro by tetradecanoyl-phorbol-13-acetate (TPA). Collagenase activity in the supernatant without and with phenylmercuric chloride activation was studied. Compared to PB PMNs, there was no consistent decrease in the total collagenase reserves in the inflammatory SF PMNs. This suggests that the release of collagenase in the inflammatory synovial fluid does not deplete SF PMNs of the collagenase synthesized at the myelocyte stage. The role of PMN collagenase in pathogenesis of cartilage destruction would then seem to be more dependent on local release and autoactivation at cartilage surface by adherent PMNs and not excessive collagenase release from free floating SF PMNs at single cell level. Furthermore, under the experimental conditions used the proportion of collagenase released in active form was higher in SF PMN specimens than in PB PMN specimens (p less than 0.01). The predominant collagenous component of adult cartilage, native type II collagen, was degraded by PMN collagenase as fast as native type I collagen. These findings suggest an important role for this enzyme in destruction of the free cartilage surface in RA.

Serum and tissue distribution of benzo[a]pyrene from intravenously injected chylomicrons in rat in vivo

Rats were injected intravenously with chylomicrons (CM) containing benzo[a]pyrene (B[a]P), a carcinogenic aromatic hydrocarbon. The disappearance of B[a]P paralleled the removal of CM, both having a rapid initial decay and a secondary slow decay. After 5 min, at the end of the rapid phase, blood contained 19% of the total injected B[a]P, with 6% in blood cells and 13% in serum. At 60 min, serum contained 5% and blood cells 6% of the total B[a]P. During the slow phase, further distribution of B[a]P within serum albumin and various lipoproteins occurred, most of the label being in low density and very low density lipoproteins. Highest tissue specific activities of [3H]B[a]P were in the lung, liver and kidney tissues. These results suggest that in the rat, distribution of ingested polycyclic aromatic hydrocarbons in vivo tends to be primarily determined by the catabolism of chylomicrons.

Helicobacter pylori infection induces a reversible expression of the CDX2 transcription factor protein in human gastric epithelium

Objective. The homeobox gene CDX2 is implicated in the appearance of intestinal metaplasia in Helicobacter pylori gastritis. The aim of this study was to investigate whether CDX2 expression in gastric mucosa occurs before the appearance of overt intestinal metaplasia in H. pylori gastritis, and whether or not this expression is reversible. Material and methods. CDX2 was studied by immunohistochemistry in a cohort of 38 patients with H. pylori gastritis before and after eradication (mean follow-up 6.3 years) of H. pylori. A cohort of 49 individuals with healthy stomachs was analysed as a control. Results. In the control group no immunostaining of CDX2 in the epithelial cells of the gastric body was found, while in 57% of the cases a mild, aberrant nuclear immunostaining of CDX2 in the non-metaplastic epithelial cells in antrum, designated as “positive staining of single cells” (PSSC), was found. In H. pylori gastritis, the PSSC was seen in antrum and corpus in 100% and 26% of the cases, respectively. The prevalence of antral PSSC was significantly increased (on average by 4-fold) in H. pylori gastritis as compared with controls. After eradication of H. pylori, the prevalence of PSSC decreased significantly in antrum but not in corpus. Conclusions. Expression of CDX2 at low intensity is common in the epithelium of normal antrum, and this expression is enhanced in H. pylori gastritis. Expression of CDX2 is reversible at least in antrum after eradication of H. pylori infection.

Early bone matrix formation during distraction: A biochemical study in sheep

Osteotomy of the distal radius and gradual distraction were performed on 20 sheep. Bone matrix synthesized between the osteotomized bone ends was biochemically characterized at 3, 5, 7, and 14 days after the start of distraction. The deposition of mineral was negligible during the 14-day period. No change was observed in the amount of organic matrix, which comprised 15 +/- 1.4 percent of the wet weight of the tissue. The amount of total protein of the wet weight in the matrix increased from 4.6 to 7.6 percent between 5 and 7 days of distraction. During the first 7 days, the nonprotein component decreased from 11 to 7.3 percent. The proportion of collagen of the total matrix protein increased gradually from 29 to 59 percent. In view of the patterns of the cyanogen bromide peptides of the matrix proteins, the heteropolymer type I[1(I)2(2)(I)1] collagen was consisted to be the principle fibrillar collagen synthesized in the distraction gap. The results suggest that tension-stress markedly affects the synthesis of fibrillar collagen towards the formation of mature bone matrix. In this process, a preliminary lag phase probably exists during which the capacity for protein synthesis is low while the synthesis of Type I collagen has started. In the secondary phase an augmented synthesis of Type I collagen takes place.

Parinaroyl and pyrenyl phospholipids as probes for the lipid surface layer of human low density lipoproteins

A simple protocol employing lipid transfer proteins was developed to label human low density lipoprotein (LDL) in a controlled manner with parinaroyl and pyrenyl phosphatidylcholines. In order to study the lipid fluidity in the surface lipid layer of LDL, the temperature-dependence of both polarization (parinaroyl probes) and excimer to monomer (E/M) intensity ratio (pyrenyl probes) were analyzed. A series of pyrenyl phosphatidylcholines containing a pyrenyl fatty acid varying from 6 to 14 carbons in length at the sn-2 position were inserted into LDL to investigate the lateral distribution of different phosphatidylcholines in the lipoprotein surface at 37 degrees C. Both polarization and E/M vs. temperature plots displayed discontinuities in the region of 22-32 degrees C, which coincides with the melting of the neutral lipid core, indicating that the latter induces an ordered to more disordered phase transition in the surface lipid layer. Determination of the E/M intensity ratio as a function of pyrene lipid concentration in LDL showed a linear relationship for the pyrenyl hexanoate and octanoate species, whereas a slope discontinuity was observed for the lipids containing a longer pyrenyl chain. These data suggest that two lipid domains with distinct properties exist in the surface layer and secondly, pyrenyl lipids partition between these domains in a chainlength-dependent manner. This is consistent with measurement of the tryptophan to pyrene energy transfer efficiency vs. pyrenyl lipid concentration, which showed a biphasic relationship for the long-chain pyrenyl lipids. These measurements further indicate that two surface lipid domains correspond to the protein-lipid boundary and the bulk lipid phase, respectively. The fact that relatively small changes in chainlength have a marked influence on the partitioning of pyrenyl lipids between the boundary and the bulk phase suggests also that native phospholipid species may not be randomly distributed in the surface lipid layer of LDL.

Correlation between the allelic distribution of STRs in a Finnish population and phenotypically different gastrointestinal tumours: a study using four X-chromosomal markers (DXS7423, DXS8377, ARA, DXS101).

Microsatellite instability in tumours has been suggested as a model to study the process of short tandem repeat (STR) mutations. In the present study we have determined the allelic variation of four X‐STRs (DXS7423, DXS8377, DXS101 and ARA) in a Finnish population of 103 individuals, and assessed whether a comparable allelic distribution could be found in a series of gastrointestinal cancers differing by the level of microsatellite instability. Fifty‐seven gastric and colorectal cancers were stratified by autosomal STRs, and the mononucleotide marker BAT‐26 into stable, low‐level unstable and high‐level unstable microsatellite (MSI‐H) cancers, of which the last produced the majority of X‐STR alleles. For the four markers analysed, a significant correlation of allele distribution between our Finnish population sample and MSI‐H tumours was noted. Together, the eight MSI‐H tumours found represented 80%, 66–80% and 100% of the DXS101 alleles in the Finnish, and in previously described Caucasian and Korean population samples, respectively. Of the ARA, DXS7423 and DXS8377 alleles in the Finnish population, 42%, 75% and 79% were found in the MSI‐H cancers, respectively. The results suggest that analysis of STR variation in a relatively small number of MSI‐H cancers may aid in pre‐evaluation of their allelic distribution in a population.

Distribution of estrogen receptors in hen oviduct chromatin fractions in the course of DNAase II digestion.

Hen oviduct chromatin was digested with DNAase II and two fractions were isolated: MgCl2-insoluble chromatin and MgCl2-soluble chromatin. The former contained 14 and 50% of the total DNA after a digestion time of 3 and 30 min, respectively. The fraction was characterized in sucrose gradients by a peak sedimenting at 11S. In the course of DNAase digestion this fraction lost most of its estrogen receptors as assayed by [3H]estradiol exchange reaction. The specific radioactivity of chromatin was particularly low in the 11S region. The MgCl2-soluble chromatin contained at most 5.1% of the total DNA. In sucrose gradients the fraction displayed peaks at 4S and 14S. After a 30 min DNAase digestion the specific radioactivity of chromatin in this fraction exceeded that of the MgCl2-insoluble fraction 7.7 fold. Material sedimenting at 14 S and at larger S values was enriched in estrogen receptors. The results suggest that estrogen receptors are unevenly distributed on hen oviduct chromain.