Two children, a girl (P1) and a boy (P2), age 5.5 and 3.5 years, respectively, with multiple fractures occurring without significant trauma, were referred for clinical eval­uation. The diagnosis of OI was based on the clinical his­tory and clinical check up, X-rays of the bones, spine and skull, biochemistry data (serum electrolytes, alkaline phosphatase), and Osteo-Sonno assessment Index (OSI) for estimation of bone density. The type of the disease was determined according to criteria provided [2].

Therapy with disodium pamidronate, 1 mg/kg body weight was applied i.v. once a month. Serum electrolytes and alkaline phosphatase were determined monthly, X-rays and bone density measurements were performed at 6-month intervals. Bone density was measured by quantita­tive ultrasound skeletal densitometry using an AOS-100 apparatus (ALOKA Company, Tokyo, Japan). The OSI was calculated for each measurement and compared with controls.

Fibroblast Cultures. Skin biopsies were taken surgi­cally 20 (P1) and 9 (P2) months after the introduction of therapy. A skin biopsy was also obtained from an unre­lated healthy child, after surgery, with the approval of the Ethics Committee of the Medical University, Bialystok, Poland, and with informed parental consent. Fibroblasts were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37°C in the presence of 5% CO₂.

Electrophoretic Analysis of Collagen. Procollagen was labeled by incubation of confluent fibroblasts with 50 mCi/mL L-[5-³H]proline (28 Ci/mmol) for 18 hours in serum-free medium containing 50 mg/mL of ascorbic acid. The procollagen was harvested separately from the cell layer and the medium in the presence of proteinase inhib­itors: 0.1 mM phenylmethanesulphonyl fluoride, 10 mM N-ethylmaleimide and 25 mM EDTA. The cell layer was scraped into 0.15 M NaCl, 50 mM Tris-HCl buffer, pH 7.5, containing the proteinase inhibitors and sonicated. Procollagens were precipitated by 25% saturated (NH₄)₂ SO₄ and the precipitates dissolved in 0.15 M NaCl, 1 mM EDTA, 50 mM Tris-HCl buffer, pH 7.5. Collagens were prepared by pepsin digestion (50 mg/mL) of procollagen samples [6]. Procollagen and collagen were electropho­resed on 5% (w/v) acrylamide separating gel with a 3.5% (w/v) acrylamide stacking gel, containing 2 M urea [6,12]. Gels were processed for fluorography with Amplify (Amersham Biosciences, Little Chalfont, Buckingham­shire, UK).

Collagen Biosynthesis. Confluent cells were labeled in serum-free medium for 18 h with the L-[5-³H]proline (5 mCi/mL, 28 Ci/mmol). Incorporation into collagen was determined by digestion of proteins with purified Clos­tridium histolyticum collagenase [13].