Hannele Uusitalo

Accelerated Turnover of Metaphyseal Trabecular Bone in Mice Overexpressing Cathepsin K

This study is based on a hypothesis that overexpression of an osteoclast enzyme, cathepsin K, causes an imbalance in bone remodeling toward bone loss. The hypothesis was tested in transgenic (TG) mice harboring additional copies of the murine cathepsin K gene (Ctsk) identifiable by a silent mutation engineered into the construct. For this study, three TG mouse lines harboring 3‐25 copies of the transgene were selected. Tissue specificity of transgene expression was determined by Northern analysis, which revealed up to 6‐fold increases in the levels of cathepsin K messenger RNA (mRNA) in calvarial and long bone samples of the three TG lines. No changes were seen in the mRNA levels of other osteoclast enzymes, indicating that the increase in cathepsin K mRNA was not a reflection of activation of all osteoclast enzymes. Immunohistochemistry confirmed that cathepsin K expression in the TG mice was confined to osteoclasts and chondroclasts. Histomorphometry revealed a significantly decreased trabecular bone volume (BV), but, surprisingly, also a marked increase in the number of osteoblasts, the rate of bone turnover, and the amount of mineralizing surface (MS). However, monitoring of bone density in the proximal tibias of the TG mice with peripheral quantitative computed tomography (pQCT) failed to reveal statistically significant changes in bone density. Similarly, no statistically significant alterations were observed in biomechanical testing at the age of 7 months. The increases in parameters of bone formation triggered by increased cathepsin K expression is an example of the tight coupling of bone resorption and formation during the bone‐remodeling cycle.

A metaphyseal defect model of the femur for studies of murine bone healing

A bone defect model was developed in the distal metaphysis of the femur for studies on bone healing in the mouse. The circular defect involving 20% of the bone circumference resulted in a 34% reduction in the bending moment compared to intact bone. The healing process was followed using histomorphometry, peripheral quantitative computed tomography (pQCT), biomechanical testing, and molecular biological analyses. Histologically, healing of the defect was characterized by filling of the medullary cavity with trabecular new bone during the first week of healing, and by closing of the cortical window by 6 weeks. Small areas of periosteal chondrogenesis were frequently observed during defect healing. In pQCT, bone mineral content (BMC) of the defect area approached that of intact control bone already by 3 weeks, reflecting the production of trabecular bone. Similarly, the bending strength and stiffness of the healing femur reached the level of intact control femur already at 3 weeks. Bone formation and remodeling was followed by Northern analyses, which demonstrated elevated mRNA levels for bone components (type I collagen and osteocalcin), and for osteoclastic enzymes (cathepsin K, matrix metalloproteinase-9, and tartrate-resistant acid phosphatase) throughout the healing period. Finally, the applicability of the defect model for gene therapy experiments was tested using adenovirus-mediated transfer of the LacZ reporter gene. Both histochemistry and mRNA analyses demonstrated that the gene was expressed in the repair tissue with the highest expression during the first week of healing. The present model thus provides a standardized environment for studies on induction and remodeling of trabecular new bone in normal and genetically engineered mice.

Expression of Cathepsins B, H, K, L, and S and Matrix Metalloproteinases 9 and 13 During Chondrocyte Hypertrophy and Endochondral Ossification in Mouse Fracture Callus

Abstract. Fracture repair provides an interesting model for chondrogenesis and osteogenesis as it recapitulates in an adult organism the same steps encountered during embryonic skeletal development and growth. The fracture callus is not only a site of rapid production of cartilage and bone, but also a site of extensive degradation of their extracellular matrices. The present study was initiated to increase our understanding of the roles of different proteolytic enzymes, cysteine cathepsins B, H, K, L, and S, and matrix metalloproteinases (MMPs) 9 and 13, during fracture repair, as this aspect of bone repair has previously received little attention. Northern analysis revealed marked upregulation of cathepsin K, MMP-9, and MMP-13 mRNAs during the first and second weeks of healing. The expression profiles of these mRNAs were similar with that of osteoclastic marker enzyme tartrate-resistant alkaline phosphatate (TRAP). The changes in the mRNA levels of cathepsins B, H, L, and S were smaller when compared with those of the other enzymes studied. Immunohistochemistry and in situ hybridization confirmed the predominant localization of cathepsin K and MMP-9 and their mRNA in osteoclasts and chondroclasts at the osteochondral junction. MMP-13 was present in osteoblasts and individual hypertrophic chondrocytes near the cartilage-bone interphase. In cartilaginous callus, the expression of cathepsins B, H, L, and S was mainly related to chondrocyte hypertrophy. During bone remodeling both osteoblasts and osteoclasts contained these cathepsins. The present data demonstrate that degradation and remodeling of extracellular matrices during fracture healing involves activation of MMP-13 production in hypertrophic chondrocytes and osteoblasts, and cathepsin K and MMP-9 production in osteoclasts and chondroclasts.

Expression Profiles of mRNAs for Osteoblast and Osteoclast Proteins as Indicators of Bone Loss in Mouse Immobilization Osteopenia Model

An experimental mouse model for disuse osteopenia was developed using unilateral cast immobilization. Analysis of the distal femurs and proximal tibias by quantitative histomorphometry revealed significant osteopenia within 10–21 days of immobilization. At 3 weeks, bone loss was also demonstrated with peripheral quantitative computed tomography as diminished bone mineral content and as concomitant reduction in the cross‐sectional moment of inertia. These structural and geometrical alterations resulted in decreased strength of the distal femurs tested by cantilever bending. Analysis of the underlying cellular and molecular mechanisms of bone loss revealed a rapid increase in bone resorption within 3 days of immobilization. The mRNA levels for cathepsin K, matrix metalloproteinase‐9, and tartrate resistant acid phosphatase were all significantly increased during the 21‐day immobilization period, but with different expression profiles. These increases were paralleled by an increased number of osteoclasts as measured by histomorphometry. By day 6 of immobilization, the balance of bone turnover was further shifted toward net bone loss as the mRNA levels for major bone components (type I collagen and osteocalcin) were decreased. In histomorphometric analysis this was observed as reduced rates of mineral apposition and bone formation after 10 days of immobilization. The results of this study demonstrate that immobilization has a dual negative effect on bone turnover involving both depressed bone formation and enhanced bone resorption.

Diminished Callus Size and Cartilage Synthesis in α1β1 Integrin-Deficient Mice during Bone Fracture Healing

Integrins mediate cell adhesion to extracellular matrix components. Integrin α1β1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in α1 integrin (α1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in α1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in α1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the α1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and α1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of α1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that α1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Accelerated up-regulation of L-Sox5, Sox6, and Sox9 by BMP-2 gene transfer during murine fracture healing.

Fracture repair is the best‐characterized situation in which activation of chondrogenesis takes place in an adult organism. To better understand the mechanisms that regulate chondrogenic differentiation of mesenchymal progenitor cells during fracture repair, we have investigated the participation of transcription factors L‐Sox5, Sox6, and Sox9 in this process. Marked up‐regulation of L‐Sox5 and Sox9 messenger RNA (mRNA) and smaller changes in Sox6 mRNA levels were observed in RNAse protection assays during early stages of callus formation, followed by up‐regulation of type II collagen production. During cartilage expansion, the colocalization of L‐Sox5, Sox6, and Sox9 by immunohistochemistry and type II collagen transcripts by in situ hybridization confirmed a close relationship of these transcription factors with the chondrocyte phenotype and cartilage production. On chondrocyte hypertrophy, production of L‐Sox5, Sox9 and type II collagen were down‐regulated markedly and that of type X collagen was up‐regulated. Finally, using adenovirus mediated bone morphogenetic protein 2 (BMP‐2) gene transfer into fracture site we showed accelerated up‐regulation of the genes for all three Sox proteins and type II collagen in fractures treated with BMP‐2 when compared with control fractures. These data suggest that L‐Sox5, Sox6, and Sox9 are involved in the activation and maintenance of chondrogenesis during fracture healing and that enhancement of chondrogenesis by BMP‐2 is mediated via an L‐Sox5/Sox6/Sox9‐dependent pathway.

Analysis of lapine cartilage matrix after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate

Objective: To study the short and long term effects of radiosynovectomy on articular cartilage in growing and mature rabbits. Methods: The articular cartilage of the distal femurs of rabbits was examined four days, two months, and one year after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate ([166Ho]FHMA). Arthritic changes were evaluated from histological sections by conventional and polarised light microscopy, and glycosaminoglycan measurements using safranin O staining, digital densitometry, and uronic acid determination. Proteoglycan synthesis was studied by metabolic [35]sulphate labelling followed by autoradiography, and electrophoretic analysis of extracted proteoglycans. Northern analyses were performed to determine the mRNA levels of type II collagen, aggrecan, and Sox9 in cartilage samples. Results: Radiosynovectomy had no major effect on the histological appearance of articular cartilage in mature rabbits, whereas more fibrillation was seen in [166Ho]FHMA radiosynovectomised knee joints of growing rabbits two months after treatment, but not after one year. Radiosynovectomy did not cause changes in the glycosaminoglycan content of cartilage or in the synthesis or chemical structure of proteoglycans. No radiosynovectomy related changes were seen in the mRNA levels of type II collagen, whereas a transient down regulation of aggrecan and Sox9 mRNA levels was seen in young rabbits two months after [166Ho]FHMA radiosynovectomy. Conclusions: [166Ho]FHMA radiosynovectomy caused no obvious chondrocyte damage or osteoarthritic changes in mature rabbits, but in growing rabbits some transient radiation induced effects were seen—for example, mild cartilage fibrillation and down regulation of cartilage-specific genes.

Induction of periosteal callus formation by bone morphogenetic protein-2 employing adenovirus-mediated gene delivery.

Although the chondrogenic response of periosteum is well established in healing fractures, the mechanisms mediating the proliferation and differentiation of periosteal chondroprogenitor cells are poorly understood. In the present study we demonstrate that bone morphogenetic protein-2 (BMP-2), introduced by adenovirus-mediated gene transfer, alone is capable of inducing callus formation at the site of periosteal injection. Both immunohistochemistry and Northern analysis demonstrated activation of type II collagen production between days 4 and 7 after the injection, followed by activation of type X collagen expression. The activation of chondrogenesis was associated with increased expression of L-Sox5 and Sox9, suggesting that the BMP-2 effect is mediated via Sox proteins. This capacity of adenovirus-mediated overproduction of BMP-2 to induce chondrogenesis (and subsequent endochondral ossification) should be useful for tissue engineering of cartilage and bone.

Production of cartilage collagens during metaphyseal bone healing in the mouse.

Small defects of unfractured bone are believed to heal without a cartilaginous intermediate. We have determined the extent of cartilage production in an experimental model of metaphyseal bone repair involving defects in both cortical and cancellous bone, but no fracture. Northern analyses revealed the presence of mRNAs for type X and II collagens in the repair tissue. Immunohistology confirmed subperiosteal deposition of both collagen types adjacent to the defect. While the mRNAs for the two collagen types peaked by one week of defect healing, immunodetectable type X collagen was not observed until the second week. The data suggest that reactivity of periosteum and activation of chondrogenesis and subsequent endochondral ossification programs are involved in murine bone repair regardless of defect type.