Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: II. Origins of Bone-Forming Cells

Two experimental models that separated demineralized bone matrix (DBM) implants from the host bone were utilized to identify the origins of bone-forming cells in the repair of calvarial defects in rats. Rat DBM, Guanadine-HCl (Gdn-HCl) extracted insoluble residue of DBM, and Gdn-HCl extracted insoluble DBM to which the dialyzed Gdn-HCl extract was added back, were implanted in the two models which prevented cells of the adjacent host bone from participating in the repair. In addition, cells in the dura and in the subcutaneous tissue overlying the calvarial defect were locally labeled with ³H-thymidine to identify the origins of those cells that were stimulated to divide and differentiate to osteoblasts. Histological studies of the temporal events that occurred during the healing process in these defect models, combined with ³H-thymidine labeling demonstrated that the osteoblasts induced by DBM were initially derived from undifferentiated mesenchymal stem cells of the dura and later augmented by cells in the overlying connective tissue covering the defect, and not from cells in the cranial bone surrounding the circular defect. The cells of both dura and subcutaneous tissue were stimulated to proliferate and differentiate principally to osteoblasts and to a very much lesser extent to chondroblasts by DBM and by reconstituted components of DBM after Gdn-HCl extraction. Gdn-HCl-extracted insoluble DBM failed to induce bone or cartilage. These results indicate that the cytokines or other factors present in DBM are required to induce bone-forming cells derived from the dura and the overlying connective tissue for the repair of the calvarial defect. Received: 1 January 1999 / Accepted: 13 August 1999     

Expression of Bone Microsomal Casein Kinase II, Bone Sialoprotein, and Osteopontin During the Repair of Calvarial Defects

The temporal expression of bone microsomal casein kinase II, osteopontin, bone sialoprotein, alkaline phosphatase, and the accumulation of a solid calcium–inorganic orthophosphate mineral phase, have been charted from day 2 to day 21 during the repair of calvarial defects in rats induced by the implantation of decalcified rat bone matrix. Unlike the sequence of events that occur when the same decalcified bone matrix is implanted subcutaneously or intramuscularly, in which cases the first tissue to form in response to the implant is cartilage that subsequently calcifies and is later resorbed and replaced by bone, the repair of cranial defects is quite different. In the latter case, the first cells induced are undifferentiated mesenchymal cells and early fibroblasts followed by osteoblastic direct bone formation. Somewhat later a few small islands of cartilage are formed, widely separated and spatially distinct from the newly formed bone matrix. All of the cartilage and most of the implanted decalcified bone matrix are later resorbed and replaced by new bone by day 21. This in vivo model of the repair of a bone defect by direct bone formation has provided an excellent system to follow specific biochemical and physicochemical events. The total accumulation and rate of accumulation of the mineral and the two noncollagenous phosphoproteins (bone sialoprotein and osteopontin), as well as the activities of alkaline phosphatase, and for the first time either in vivo or in cell culture, the activity of microsomal casein kinase II, the major enzyme that phosphorylates the bone phosphoproteins, have been determined as a function of healing time in vivo. The overall general pattern of accumulation of the phosphoproteins and calcium-phosphate mineral phase and their relationships are similar to those reported in osteoblast cell cultures also monitored as a function of time.     

Bone Morphogenetic Protein-2 Increases the Rate of Callus Formation after Fracture of the Rabbit Tibia

The effects of human recombinant bone morphogenetic protein-2 (rhBMP-2) on rabbit fractures healing under both stable and unstable mechanical conditions were investigated. rhBMP-2 was administered (1) on bioerodible particles, (2) in a collagen gel, and (3) by injection. rhBMP-2 on bioerodible particles has no effect as the particles prevent the migration of cells that produce the callus. The collagen gel is resorbed more rapidly; the development of the callus of mechanically unstable fractures is similar to controls at 14 days. When rhBMP-2 is injected, the callus of mechanically unstable fractures develops more rapidly so that cortical union occurs by 21 days, as compared with 28 days in control fractures. The effects on fractures healing under stable mechanical conditions are minimal. It is argued that mechanical factors influence the size of the callus of normally healing fractures and, although BMP-2 accelerates the rate of development of the callus and cortical union, it does not affect the amounts of bone and cartilage produced. Received: 9 February 1998 / Accepted: 9 December 1998     

Insulin-like Growth Factor I Does Not Stimulate Bone Resorption in Cultured Neonatal Mouse Calvarial Bones

Insulin-like growth factor I (IGF-I) has documented anabolic effects on osteoblasts, whereas its influence on osteoclasts and on bone resorption is unclear. We have investigated the effects of IGF-I on osteoclast recruitment and bone resorption in vitro. IGF-I (at and above 1 nM) stimulated the formation of multinucleated tartrate-resistant acid phosphatase positive cells in murine bone marrow cultures, incubated for 9 days. The number of multinucleated cells increased to 540 ± 160% of control (mean ± SEM) in cultures treated with 10 nM IGF-I. IGF-I (0.1–100 nM) had no effect by itself on ⁴⁵Ca-release from prelabelled neonatal mouse calvarial bones. However, IGF-I (100 nM) had an inhibitory effect on bone resorption induced by prostaglandin E₂ and 1,25(OH)₂D₃. These findings indicate that IGF-I enhances the formation of osteoclasts-like cells in long-term bone marrow cultures. In bone organ cultures, however, IGF-I has an inhibitory effect on stimulated bone resorption, suggesting that IGF-I inhibits existing osteoclasts and, alternatively, that IGF-I interferes with the osteoblast-derived factor(s) that stimulate existing osteoclasts. Received: 15 August 1995 / Accepted: 1 April 1996     

Mitogenic Lectin Concanavalin A Induces Calvarial Bone Formation In Vivo via Indomethacin-Sensitive Pathway

Natural products such as plant lectins have not been extensively surveyed for their potential as anabolic agents. Wlodarski (1991) observed that the lectin Concanavalin A (ConA) has chondrogenic and osteogenic activity following local injection over the mouse tibia. To gain more insight into the mechanism of ConA in bone, we investigated and quantitated the effects of ConA injected locally over the mouse calvaria in vivo. ConA was injected subcutaneously over the calvaria of mice at 2, 10, and 20 μg per injection, four times a day, for three consecutive days. By day fourteen, a layer of new woven bone 30 μm thick had been laid down on the periosteal surface, resulting in a 36% increase in calvarial thickness (as compared with 2% in vehicle-treated controls). ConA also increased periosteal width and osteoblast surface in a dose-dependent manner. Concurrent administration of indomethacin (30 μg) with ConA (20 μg), four times a day for 3 days, strongly inhibited new bone formation. With a single injection of ConA (80 μg) over the calvaria, osteoclastic bone resorption and proliferation of osteoblast precursors and periosteum increased at day four, but showed a decrease at later times (14 and 28 days after injection). Except at the earliest time, there was little evidence of osteoclastic bone resorption. New bone width increased linearly over 28 days. In summary, ConA induced new bone formation in a pattern comparable with that of aFGF and bFGF, potent stimulators of calvarial bone formation (Dunstan, 1993), and this osteogenic effect was caused by an indomethacin-sensitive pathway. Received: 15 January 1996 / Accepted: 11 April 1996     

Bone Mineral Density and Bone Turnover in Patients with Knee Osteoarthritis Compared with Generalized Osteoarthritis

The aim of this study was to investigate bone mineral density (BMD) and bone turnover in patients with primary knee osteoarthritis (KOA) and to compare them with generalized OA (GOA) and nonGOA patients. A total of 88 postmenopausal primary KOA patients were studied. OA was graded by using knee radiographs. BMD of the lumber spine, femur, and radius, and biochemical markers of bone turnover, pyridinoline (Pyr), deoxypyridinoline (Dpyr), CTx, and osteocalcin were compared among each grade. BMD was also compared with 88 normal controls who were age and weight-matched. In 88 KOA patients, 56 were divided into 28 GOA and 28 non-GOA groups by grading hand radiographs. BMD and biochemical markers were compared between GOA and non-GOA. KOA patients had higher BMD at several skeletal sites compared with age- and weight-matched normals. A significant difference of BMD between each grade was observed between grades 0–1 and 3 (0.774 ± 0.143 versus 0.940 ± 0.185 g/cm², P < 0.001), grades 2 and 3 (0.781 ± 0.125 versus 0.940 ± 0.185 g/cm², P < 0.01) in the spine, and between grades 0–1 and 3 (0.505 ± 0.100 versus 0.564 ± 0.127 g/cm², P < 0.05) in the trochanter. A significant difference of biochemical bone markers was observed between grades 0–1 and 3 (P < 0.05) and between grades 2 and 3 (P < 0.05) in Pyr and grades 0–1 and 3 (P < 0.05) and between grades 1 and 4 (P < 0.05) in Dpyr, but not in osteocalcin and CTx. GOA patients had higher BMD of the spine (0.902 ± 0.175 versus 0.747 ± 0.138 g/cm², P < 0.01), trochanter (0.535 ± 0.107 versus 0.480 ± 0.107 g/cm², P < 0.05), and one-third of the radius (0.526 ± 0.068 versus 0.472 ± 0.089 g/cm², P < 0.05) and had significantly higher biochemical markers in Pyr and Dpyr than non-GOA patients. It is concluded that KOA patients had higher BMD at several skeletal sites. Biochemical bone markers were influenced by some degree of cartilage damage in OA patients. This tendency was stronger in GOA patients than in non-GOA patients. Received: 12 February 1999 / Accepted: 2 November 1999     

骨髓基质细胞修复颅骨缺损的实验研究

目的为验证三维打印研究中动物模型的可靠性，采用骨髓基质干细胞（Bone marrow mesenchymal stem cells，BMSCs）修复裸鼠颅骨缺损。方法共10只裸鼠，其中实验组（n=5只）：颅骨缺损区植入BMSCs／脱钙骨；材料对照组：颅骨缺损区植入单纯脱钙骨，采用实验组自身对照（n=5只）；空白对照组（n=5只）：双侧颅骨区造成缺损后旷置。术后3个月取材进行大体观察、组织学和免疫组化（骨钙蛋白）检查。结果术后3个月，实验侧组织质地较硬，组织学表现为骨结构，骨钙蛋白免疫组化阳性；对照侧质地较软，组织学表现为结缔组织，骨钙蛋白免疫组化阴性；空白对照组仍表现为颅骨缺损。结论采用BMSCs可修复裸鼠颅骨缺损，验证了三维打印研究中动物模型的可靠性。     

Influence of bone‐derived matrices on generation of bone in an ectopic rat model

Most bone regeneration experimental models that test bone‐derived matrices take place in conjunction with the native bone. Here, we compared the relative effectiveness of bone matrix components on bone‐marrow‐directed osteogenesis in an ectopic model. Cortical bone cylinders consisted of diaphysis of DA rat femurs. They were either demineralized (DBM), deproteinized (HABM), or nontreated (MBM). Fresh bone marrow was placed into cylinders and implanted at subcutaneous thoracic sites of 2‐month‐old DA rats. At designated times the cylinders were surgically removed from the animals. Microradiographs of DBM and histology of DBM and MBM cylinders demonstrated progressive increase in mineralized bone volume and its trabecular configuration. Bone filled the inner volume of DBM and MBM cylinders within 4 weeks, while in HABM cylinders mostly granulation tissue developed. In the DBM cylinders cartilage deposited within 10 days, while in the MBM cylinders bone was directly deposited. As early as day 3 after marrow transplantation, marrow cells interacting with DBM increased significantly the genes that express the cartilage and the bone phenotype. In conclusion, organic components of bone are needed for marrow‐directed osteogenesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:664–670, 2010     

Bone repair induced by bone morphogenetic protein in ulnar defects in dogs

In dogs, resection of a length of the ulna equal to twice the diameter of the mid-shaft leaves a defect which consistently fails to unite. In response to an implant of 100 mg of bovine bone morphogenetic protein (BMP), the defect becomes filled by callus consisting of fibrocartilage, cartilage and woven bone within four weeks. The cartilage is resorbed and replaced by new bone in four to eight weeks. Woven bone is then resorbed, colonised by bone marrow cells and remodelled into lamellar bone. Union of the defect is produced by 12 weeks. Control defects filled with autogeneic cortical bone chips unite after the same period. In regeneration induced by bone morphogenetic protein (BMP) and in repair enhanced by bone graft, union depends upon the proliferation of cells within and around the bone ends. Our working hypothesis is that BMP induces the differentiation of perivascular connective tissue cells into chondroblasts and osteoprogenitor cells and thereby augments the process of bone regeneration from the cells already present in the endosteum and periosteum.     

Different Behavior of Human Osteoblast-Like Cells Isolated from Normal and Heterotopic Bone In Vitro

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM β-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3–6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells. Received: 29 March 1996 / Accepted: 21 May 1997     

同种异体脂肪干细胞复合脱钙骨修复大鼠尺骨缺损

目的探讨同种异体脂肪干细胞修复管状骨缺损的可行性。方法获取SD大鼠的腹股沟处脂肪,分离培养脂肪干细胞（Adipose-Derived Stem Cells,ADSCs）;鼠第3代ADSCs与脱钙骨复合,24 h后进行成骨诱导培养。检测细胞在材料表面的生长及成骨分化能力。建立鼠两侧尺骨缺损模型,分别植入鼠ADSCs-脱钙骨复合物（实验侧）和单纯脱钙骨材料（对照侧）;8周、24周后取样,行DR和组织学检测,观察成骨情况。结果 ADSCs能在脱钙骨上很好地黏附和生长,并维持成骨分化能力。细胞-材料复合物植入24周后,DR显示实验侧有新生骨基质长成,对照侧未见骨组织生成。组织学检测显示,实验侧缺损区被典型的骨组织取代,可见新生骨小梁附着于脱钙骨表面;对照侧只有少量的骨组织和纤维组织充填。结论 ADSCs-脱钙骨材料复合物植入,能成功修复临界大小的管状骨缺损。     

The Role of Chondrocytes in Intramembranous and Endochondral Ossification During Distraction Osteogenesis in the Rabbit

We have used a rabbit leg-lengthening model for detailed studies of the histology of distraction osteogenesis. Some unusual features of the endochondral ossification that occurs during the rapid transition of cartilage to bone in the regenerate were observed. Histological staining techniques together with immunohistochemistry and nonradioactive in situ mRNA hybridization for cartilage and bone-related molecules have been used to document the presence of an overlapping cartilage-bone phenotype in cells of the cartilage-bone transitional region. In those particular areas, some chondrocytes appeared to be directly transformed into newly formed bone trabeculae which are surrounded by bone matrix. Acid phosphatases were found within the cartilage matrix in some of the cartilage/bone transitional regions and type I collagen mRNA and type II collagen protein were found together in some of the marginal hypertrophic chondrocytes. This study indicates an unusual role of chondrocytes in the process of ossification at a distraction rate of 1.3 mm/day in the rabbit. Further direct evidence is required to prove the hypothesis that the hypertrophic chondrocytes may transdifferentiate into bone cells in this model. Received: 13 March 1997 / Accepted: 22 September 1998     

Local Application of Basic Fibroblast Growth Factor Minipellet Induces the Healing of Segmental Bony Defects in Rabbits

Fibroblast growth factor (FGF) has been reported to increase the volume of callus in a fracture model of rats. There are, however, no reports of successful repair of segmental bony defects by application of an FGF solution. In this study, the effects of basic FGF on the repair of segmental bony defects in the rabbit femur were examined. Minipellet, a new drug delivery system using atelocollagen, was employed to ensure effective delivery of FGF. Segmental bony defects (10 mm in length) were created in the right femurs of 19 rabbits. In pilot studies, no defects of this size healed spontaneously within 6 weeks. Bones were stabilized with miniexternal fixators. Minipellets containing basic FGF were implanted between fragments so as to bridge the two fragments. The healing processes were monitored radiographically and studied histologically. In rabbits in which FGF was added to the defect site at doses of 1.4 μg or higher, approximately 90% of the defects were filled with new bone and cartilage within 6 weeks after minipellet implantation. In rabbits receiving placebo minipellets, however, approximately 15% of the defects were filled by callus within 6 weeks. Furthermore, this callus did not change into mature bone. An injection of 2 μg of FGF solution to bony defects had no effect on the repair of segmental bony defects. These findings suggest that FGF plays a role in the production of adequate volumes of callus particularly in the initial stages of fracture healing and that sustained local release enables FGF to be effective at a low dose. In summary, large segmental bony defects healed after insertion of low-dose FGF minipellets. An adequate dose of FGF and an appropriate delivery system are required for successful healing of large bony defects. These findings imply the potential value of FGF minipellets in clinical practice. Received: 26 June 1997 / Accepted: 11 May 1998     

自体骨髓基质干细胞组织工程骨修复颅骨缺损的临床研究

目的探讨人骨髓基质干细胞(hBMSCs)作为种子细胞的组织工程骨修复外伤后颅骨缺损的可行性。方法自2006年6月至2007年2月,共4例外伤后颅骨缺损患者,抽取患者骨髓,分离得到hBMSCs,体外扩增和成骨诱导后,将hBMSCs与部分脱钙骨复合,体外共培养1周后,手术植入颅骨缺损区。分别于术后1周和3个月、6个月进行临床和三维CT检查随访。结果术后1周,三维CT均显示骨缺损区被所植入的组织工程骨充填;术后3～6个月,CT显示组织工程骨形成并修复骨缺损,新生骨与骨缺损断端融合。高龄患者及骨缺损面积过大患者,组织工程骨体内成活率较差。结论通过选择适宜的病例,以自体hBMSCs作为种子细胞,运用组织工程技术可以在人体内形成稳定的组织工程骨并可用于修复颅骨缺损。     

Effects of Ipriflavone on Perialveolar Bone Formation

The effect of ipriflavone (IP), a synthetic isoflavonoid derivative, on in vivo bone formation was studied in rat perialveolar bone by surgically producing a hole in the mandibular bone. The holes were filled either with powdered IP or with compounds containing no osteoinductive properties such as biostite and Htr (hard tissue replacement). In control animals, the holes were left to heal spontaneously. The animals were killed 3, 28, and 40 days after surgery and a detailed morphological and morphometric study was performed on the perialveolar bone surrounding the wounds. Three days after surgery (inflammatory phase) the bone wounds were occupied by hemorragic and inflammatory cells in both the untreated and IP-treated bone defects. Twenty-eight days after surgery, bone formation was evident with new bone spiculae particularly concentrated in the area of the bone lesion closest to the adjacent periodontal ligament. Morphometric measurements of the areas occupied by new bone showed that the synthesis of perialveolar bone was significantly stimulated by IP. The repair of the bone defects by new bone formation progressed by day 40, but only in the presence of IP were the original holes almost completely repaired. Conversely, biostite and Htr did not influence promotion of new bone formation. In conclusion, the results of the present study are consistent with a role of IP in stimulating osteogenesis and suggest that this compound could represent a potential therapeutic tool to promote repair of injured perialveolar bone.     

Biochemical Markers of Bone Turnover Reflect Femoral Bone Loss in Elderly Women

Although over 90% of hip fractures occur in patients over age 70, few data are available on femoral bone loss in this age group. To examine the relationship between biochemical markers of bone turnover and femoral bone loss in the elderly, 36 female and 17 male, healthy, community-dwelling elderly over age 65 (mean ± SD age: women 71 ± 4 years, men 75 ± 5 years) were followed for 3 years. Annual bone mineral density measurements of the hip and lumbar spine by dual-energy x-ray absorptiometry (DXA) were obtained and biochemical markers of bone resorption (urinary N-telopeptide crosslinks, free pyridinoline, total pyridinoline, total deoxypyridinoline, and hydroxyproline) and bone formation (serum osteocalcin, bone-specific alkaline phosphatase) were obtained at the end of year 3. In elderly women, longitudinal bone loss at the total hip was negatively correlated with markers of bone resorption (r =−0.39 to −0.52, P < 0.05), bone formation (r =−0.38, P < 0.05), and age (r =−0.39, P < 0.05). Markers of bone resorption were correlated with markers of bone formation (r = 0.63 to 0.74, P < 0.01). In multiple regression analysis, urinary N-telopeptide crosslinks (marker of resorption), serum osteocalcin (marker of formation), and serum parathyroid hormone explained 43% of the variability of bone loss at the total hip in women. These parameters were not related to bone loss in men. We conclude that femoral bone loss increases with age in women over 65. Measurements of specific biochemical markers of bone turnover are correlated with longitudinal bone loss in elderly women. These markers may help identify women at greatest risk for bone loss who would benefit most from therapeutic interventions. Received: 28 January 1996 / Accepted: 3 May 1996     

Osteogenesis Imperfecta: Bone Turnover, Bone Density, and Ultrasound Parameters

We studied 21 patients (11 men and 10 women) with osteogenesis imperfecta (OI) and 21 age- and sex-matched controls. In all patients we measured serum levels of total alkaline phosphatase (ALP), type I procollagen carboxy-terminal propeptide (PICP), osteocalcin (BGP), urinary excretion of hydroxyproline (HOP/Cr), and pyridinoline crosslinks (Pyr/Cr). Bone mineral density was measured at the distal radius (BMD-R) and at the lumbar spine (BMD-LS) by dual X-ray absorptiometry (DXA). Ultrasound parameters were also performed at the calcaneous with the Achilles device and at the phalanxes with DBM Sonic 1200. A significant reduction (P < 0.001) in BMD and in ultrasound parameters was found in OI patients compared with normals. PICP was significantly reduced in the OI patients compared with controls (P < 0.001); other markers of bone turnover were higher in OI than in controls, but the difference did not reach the statistical significance. A significant correlation (P < 0.05) was found between PICP and BMD at the lumbar spine and between PICP and ultrasound parameters at the calcaneous. On the basis of our data, we conclude that patients with OI show low values of BMD and ultrasound parameters; therefore in these patients, not only is bone mass disturbed but also bone quality. The reduced levels of PICP in OI patients confirm that most OI patients have defects in collagen I biosynthesis. These defects may contribute to the fragility of OI bone by interfering with complete mineralization and/or normal tissue structure. PICP may be considered a useful marker in the clinical management of OI. Received: 26 March 1998 / Accepted: 15 January 1999     

Gonadal Steroids and Bone Metabolism in Young Castrated Male Rats

At 45 days of age, 40 male Wistar rats were castrated, then randomly divided into four groups, S.C. injected for 60 days after surgery either with 17β-estradiol (E) 10 μg/kg BW/48 hours, progesterone (P) 140 μg/kg BW/48 hours, dihydrotestosterone (D) 2 μg/kg BW/48 hours, E + P + D same doses, or solvent alone (CX). Ten other rats were sham-operated (SH) and used as controls. Animals were put in balance to determine Ca and phosphorus (Pi) intestinal apparent absorption (IA Ca, IA Pi) and urinary pyridinium crosslinks excretion. Plasma was collected for measurement of intact-parathyroid hormone (PTH), calcitonin (CT), insulin-like growth factor I (IGF-I), 1,25 dihydroxyvitamin D (1,25(OH)₂D), Ca, and Pi. Orchidectomy induced marked seminal vesicles atrophy and increased plasma CT, PTH, and Ca concentrations. IA Ca was significantly higher in P rats, however, neither castration nor any other treatment had significant effects. Orchidectomy decreased femoral length, dry weight, and Ca content, whereas E or D given alone or together with P improved endochondral growth and enhanced femoral Ca content. Again, bone mineral density was lowered by orchidectomy and reestablished by both E and EPD, even above SH values, this effect being more important at the metaphyseal levels. Urinary pyridinium cross-links excretion and plasma osteocalcin concentrations were higher in the CX animals than in the controls. Although E and D given alone did reduce both biochemical turnover markers, they showed additive effect when given together (EPD). In conclusion, in the young castrated male rat, E was more efficient than D for preventing bone loss, the most important effect being induced by a combination of E + P + D. Received: 28 June 1999 / Accepted: 12 January 2000