Matti Kallioinen

MATRIX METALLOPROTEINASE‐2 (72 kD TYPE IV COLLAGENASE) EXPRESSION OCCURS IN THE EARLY STAGE OF HUMAN MELANOCYTIC TUMOUR PROGRESSION AND MAY HAVE PROGNOSTIC VALUE

Matrix metalloproteinase‐2 (MMP‐2), a member of the matrix metalloproteinase family, participates in degradation of the pericellular and extracellular matrix during neoplastic growth and metastasis. Experimental data have substantiated its role in melanoma invasion, but there is no information at present concerning its expression in histological specimens from human melanocytic tumours. This study describes the occurrence and immunolocalization of MMP‐2 in human melanocytic lesions, defining distinct steps in melanoma progression. Paraffin‐embedded sections from 118 melanocytic lesions were immunostained using a specific antibody to 72 kD type IV collagenase. The material included 34 common naevocellular naevi, 14 dysplastic naevi, 21 in situ melanomas, 20 primary malignant melanomas, and 29 melanoma metastases. Intracytoplasmic MMP‐2 immunoreactive protein was found in the ‘naevocytic nests’ of common naevi, in junctional naevus cells, and in melanoma cells. The surrounding normal skin stained negatively, except for occasional macrophages, sweat glands, and hair follicles. The number of MMP‐2‐positive cells increased with decreasing architectural organization and increasing atypia in the melanocytic lesions. The MMP‐2 positivity in the primary and subcutaneous melanoma lesions correlated with later haematogenous metastasis. The data suggest that MMP‐2 expression is an early event in melanocytic tumour progression, but is nevertheless prognostic for haematogenous metastasis in melanoma.

Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP‐2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP‐2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP‐2 as a possible prognostic marker in melanoma. The expression of MMP‐2 immunoreactive protein was studied with immunoperoxidase staining in paraffin‐embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty‐four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP‐2 in tumour cells. Marked overexpression of MMP‐2 protein (≥34 per cent of melanoma cells positive) correlated with the 5‐year survival of the patients when compared with patients with lower MMP‐2 positivity, 55 per cent vs. 85 per cent, respectively (P<0·05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP‐2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10‐year survival for MMP‐2‐positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P<0·05). In the hierarchic Cox regression model for survival, MMP‐2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP‐2 protein indicated a 4·5‐fold relative risk of dying from melanoma. It is concluded that MMP‐2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP‐2 expression in male melanoma patients indicates an unfavourable prognosis.

Smoking affects collagen synthesis and extracellular matrix turnover in human skin

Summary Background Smoking is associated with premature facial wrinkling and aberrant wound healing, but the underlying mechanisms of skin injury are poorly understood.

Bone healing and mineralization, implant corrosion, and trace metals after nickel–titanium shape memory metal intramedullary fixation

Its shape memory effect, superelasticity, and good wear and damping properties make the NiTi shape memory alloy a material with fascinating potential for orthopedic surgery. It provides a possibility for making self-locking, self-expanding, and self-compressing implants. Problems, however, may arise because of its high nickel content. The purpose of this work was to determine the corrosion of NiTi in vivo and to evaluate the possible deleterious effects of NiTi on osteotomy healing, bone mineralization, and the remodeling response. Femoral osteotomies of 40 rats were fixed with either NiTi or stainless steel (StSt) intramedullary nails. The rats were killed at 2, 4, 8, 12, 26, and 60 weeks. Bone healing was examined with radiographs, peripheral quantitative computed tomography, (pQCT) and histologically. The corrosion of the retrieved implants was analyzed by electron microscopy (FESEM). Trace metals from several organs were determined by graphite furnace atomic absorption spectrometry (GF-AAS) or by inductively coupled plasma-atomic emission spectrometry (ICP-AES). There were more healed bone unions in the NiTi than in the StSt group at early (4 and 8 weeks) time points. Callus size was equal between the groups. The total and cortical bone mineral densities did not differ between the NiTi and StSt groups. Mineral density in both groups was lower in the osteotomy area than in the other areas along the nail. Density in the nail area was lower than in the proximal part of the operated femur or the contralateral femur. Bone contact to NiTi was close. A peri-implant lamellar bone sheet formed in the metaphyseal area after 8 weeks, indicating good tissue tolerance. The FESEM assessment showed surface corrosion changes to be more evident in the StSt implants. There were no statistically significant differences in nickel concentration between the NiTi and StSt groups in any of the organs. NiTi appears to be an appropriate material for further intramedullary use because it has good biocompatibility in bone tissue.

Bone modeling and cell–material interface responses induced by nickel–titanium shape memory alloy after periosteal implantation

The purpose of this study was to evaluate the new bone formation, modeling and cell-material interface responses induced by nickel-titanium shape memory alloy after periosteal implantation. We used a regional acceleratory phenomenon (RAP) model, in which a periosteal contact stimulus provokes an adaptive modelling response. NiTi has thermal shape memory and superelasticity properties uncommon in other implant alloys. So far, there are insufficient data concerning the biocompatibility of NiTi as a bone implant. NiTi was compared to stainless steel (stst) and Ti-6Al-4V. The test implant was placed in contact with the intact femur periosteum, but it was not fixed inside the bone. Histomorphometry with digital image analysis was used to determine the bone formation and resorption parameters. The ultrastructural features of cell-material adhesion were analysed with scanning electron microscopy (FESEM). A typical peri-implant bone wall modelation was seen due to the normal RAP. The maximum new woven bone formation started earlier (2 weeks) in the Ti-6Al-4V group than in the NiTi (P < 0.01) group, but also decreased earlier, and at 8 weeks the NiTi (P < 0.05) and stst (P < 0.005) groups had greater cortical bone width. At 12 and 26 weeks no statistical differences were seen in the histomorphometric values. The histological response of the soft tissues around the NiTi implant was also clearly non-toxic and non-irritating. Cell adhesion and focal contacts were similar between the materials studied by FESEM. We conclude that NiTi had no negative effect on total new bone formation or normal RAP after periosteal implantation during a 26-week follow-up.

Matrix metalloproteinase‐2 (MMP‐2) immunoreactive protein—a new prognostic marker in uveal melanoma?

Uveal melanoma is the most common primary intraocular tumour. Once haematogenous metastasis has occurred, there is no cure for the disease and there is an obvious need for new biological prognostic markers to estimate the risk of metastasis. In this study, the expression of matrix metalloproteinase‐2 (MMP‐2) was characterized immunohistochemically in 29 human uveal melanomas. Enzyme‐linked immunoassays and gelatin zymographies were assessed in order to quantify the expression of gelatinases A and B, as well as the tissue inhibitor of metalloproteinases (TIMPs), in the vitreous body. A total of 49 per cent of the uveal melanomas displayed a positive immunoreaction for MMP‐2 in melanoma cells, the epithelioid cells showing the most frequent staining. There was no correlation between the positivity of MMP‐2 staining and the size of the primary tumour, gender or age. The expression of MMP‐2 was associated with a dismal prognosis: the 5‐year overall survival rate for MMP‐2‐positive cases was significantly inferior to that of the MMP‐2 negative cases, 49 per cent vs. 86 per cent, respectively (p=0·02). A patient group at high risk of metastatic disease was identified; only 38 per cent of patients with a MMP‐2‐positive non‐spindle cell uveal melanoma survived for 5 years. The analyses of MMPs or TIMPs in the vitreous body had no prognostic value. Positive immunostaining for MMP‐2 was observed in the retinal pigment epithelium, corneal epithelium, and fibroblasts in the ciliary body and choroid. It is concluded that immunohistochemical analysis of MMP‐2 may help to predict a risk of metastasis in uveal melanoma. Copyright

Systemic therapy with estrogen or estrogen with progestin has no effect on skin collagen in postmenopausal women

OBJECTIVES To investigate the effect of estrogen alone or combined with progestin on the amount and synthesis of skin collagen in postmenopausal women. METHODS Forty-three early postmenopausal women were enrolled into this open, non-randomized parallel-groups study. Fifteen women received a continuous oral dose of 2 mg of 17 beta-estradiol and 1 mg of norethisterone acetate daily and 14 women an oral dose of 2 mg estradiol valerate daily. Fourteen subjects served as controls. The histology and type I and III procollagen immunohistochemistry of the skin, skin thickness, the amount of total collagen determined by a colorimetric method and the synthesis of type I and III collagens determined by analysing procollagen propeptides in the suction blister fluid were studied before the treatment and at 6 and 12 months. The proportional area of elastic fibers and the thickness of the epidermis were assessed from the sections obtained before the treatment and at 12 months, with computerized image analysis. RESULTS Skin thickness, the amount and rate of collagen synthesis, the proportional area of elastic fibers and the thickness of the epidermis were not affected by either 17 beta-estradiol and 1 mg of norethisterone acetate or 2 mg of estradiol valerate. No histological or immunohistological changes were detected in the skin specimens during the 12-month treatment period compared to the baseline or to the skin specimens of the control group. CONCLUSIONS A 1-year treatment with systemic estrogen alone or combined with progestin does not change the amount of collagen or the rate of collagen synthesis in postmenopausal women.

Biocompatibility and strength properties of nitinol shape memory alloy suture in rabbit tendon.

Nitinol (NiTi) is a promising new tendon suture material with good strength, easy handling and good super-elastic properties. NiTi sutures were implanted for biocompatibility testing into the right medial gastrocnemius tendon in 15 rabbits for 2, 6 and 12 weeks. Additional sutures were implanted in subcutaneous tissue for strength measurements in order to determine the effect of implantation on strength properties of NiTi suture material. Braided polyester sutures (Ethibond) of approximately the same diameter were used as control. Encapsulating membrane formation around the sutures was minimal in the case of both materials. The breaking load of NiTi was significantly greater compared to braided polyester. Implantation did not affect the strength properties of either material.

P53 Accumulation, Deoxyribonucleic Acid Ploidy and Progression of Bladder Cancer

PURPOSE The alterations in deoxyribonucleic acid (DNA) ploidy and p53 expression during progression of bladder cancer were determined. MATERIALS AND METHODS p53 Expression and DNA ploidy were studied in 51 patients with transitional cell carcinoma of the bladder (mean followup 5 years). Of 29 primarily superficial tumors (stages Ta and T1) 17 became subsequently invasive (greater than stage T2) within an average of 4 years (group 2) and 12 recurred superficially with no sign of progression during a mean followup of 10.8 years (group 1). Of the patients 22 had metastatic disease (group 3). Samples of tumors at diagnosis and recurrence or progression were analyzed by immunohistochemistry and flow cytometry. RESULTS p53 Accumulation was detected at diagnosis in 29 of the 51 patients (57%), including 3 of 12 (25%) in group 1, 5 of 17 (29%) in group 2 and 14 of 22 (64%) in group 3. The p values for the differences between groups 1 and 3, and 2 and 3 were 0.07 and 0.054, respectively. Abnormal DNA contents were noted in 3 of 12 (25%), 11 of 17 (65%) and 16 of 22 (73%) patients in groups 1 to 3, respectively, and the differences between groups 1 and 2, and 1 and 3 were statistically significant. Using these 2 genetic markers, we found genetic progression to be uncommon in groups 1 and 3, whereas in group 2 an initially negative p53 staining became positive at invasion into the muscle in 5 of 12 patients (42%). CONCLUSIONS The tumors in patients with superficial recurrences are mostly diploid and negative for p53, and those with metastasis are nondiploid and positive for p53 from the beginning, while further genetic progression is uncommon. However, p53 tends to accumulate frequently when the tumor begins to invade the muscle. There seems to be a need for caution against under staging an apparent stage T1 tumor that is positive for p53.

Expression of cytokeratins in regenerating human epidermis

The expression of cytokeratin polypeptides in regenerating human epidermis was immunohistochemically examined during re‐epithlization of suction blisters. The regenerating basal and suprabasal epidermis expressed keratin polypeptides K13, K14, K16 and K18, which are not present in normal suprabasal epidermis. On the contrary, K10, a normal constituent of terminally differentiated keratinocytes, was lacking from the epidermis until the ninth day of re‐epithelization. The findings indicate changes similar to other hyperproliferative states (expression of K16), basal‐like features (expression of K14), or properties reminiscent of fetal skin (K13 and K18) in the newly formed epidermis. Monoclonal antibodies for cytokeratins and a technique using suction blisters seemed to be suitable methodology for the study of epidermal regeneration in normal skin. The technique may also advantages in the investigation of keratin expression in diseased skin.