Intracellular tension in periosteum/perichondrium cells regulates long bone growth

Perichondrium/periosteum is involved in regulating long bone growth. Long bones grow faster after removal or circumferential division of periosteum. This can be countered by culturing them in conditioned medium from perichondrium/periosteum cells. Because both complete removal and circumferential division are effective, we hypothesized that perichondrium/periosteum cells require an intact environment to release the appropriate soluble factors. More specifically, we propose that this release depends on their ability to generate intracellular tension. This hypothesis was explored by modulating the ability of perichondrium/periosteum cells to generate intracellular tension and monitoring the effect thereof on long bone growth. Perichondrium/periosteum cells were cultured on substrates with different stiffness. The medium produced by these cultures was added to embryonic chick tibiotarsi from which perichondrium/periosteum was either stripped or left intact. After 3 culture days, long bone growth was proportionally related to the stiffness of the substrate on which perichondrium/periosteum cells were grown while they produced conditioned medium. A second set of experiments demonstrated that the effect occurred through expression of a growth‐inhibiting factor, rather than through the reduction of a stimulatory factor. Finally, evidence for the importance of intracellular tension was obtained by showing that the inhibitory effect was abolished when perichondrium/periosteum cells were treated with cytochalasin D, which disrupts the actin microfilaments. Thus, we concluded that modulation of long bone growth occurs through release of soluble inhibitors by perichondrium/periosteum cells, and that the ability of cells to develop intracellular tension through their actin microfilaments is at the base of this mechano‐regulated control pathway. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:84–91, 2011     

Organization and cellular biology of the perichondrial ossification groove of ranvier: a morphological study in rabbits.

The perichondrial ossification groove of Ranvier, a circumferential groove in the periphery of the epiphyseal cartilage, was studied in rabbits whose ages ranged from one week to eight months using light and electron microscopy, autoradiography after labeling with 3H-thymidine, 3H-proline, and 3H-glucosamine, and histochemical staining for proteoglycans and alkaline phosphatase. By these methods, three groups of cells were identified within the groove: 1. A group of densely packed cells deep in the groove, which are the progenitor cells for the osteoblasts that form the bone bark, a cuff of bone surrounding the epiphyseal growth-plate region and the adjacent part of the metaphysis. 2. A group of more widely dispersed, relatively undifferentiated mesenchymal cells and fibroblasts, some of which are chondroblast precursors that probably contribute to appositional chondrogenesis and growth in width of the epiphyseal cartilage. 3. Fibroblasts and fibrocytes among sheets of highly oriented and organized collagen fibers which form a fibrous layer that is continuous with the outer fibrous layer of the periosteum and with the perichondrium. This layer also sends fibers into the epiphyseal cartilage and anchors the periosteum firmly to the epiphyses as bone growth proceeds.     

Changes of elastic fibers in musculoskeletal tissues of Marfan syndrome: a possible mechanism of joint laxity and skeletal overgrowth.

The aim of the study was to analyze by histochemical, ultrastructural, and morphometric methods the musculoskeletal tissues in three humans affected with Marfan syndrome. Histochemical and morphometric data demonstrated that the content of elastic fibers in the perichondrium, periosteum, and knee capsule of the individuals with Marfan syndrome was dramatically reduced in comparison with control tissues. Ultrastructurally the elastic fibers appeared fragmented and indented, because of the presence of discontinuous aggregates of elastin among randomly dispersed filaments. These abnormalities of the articular capsule argue that these fibers could be functionally incompetent to resist normal stress, predisposing to joint laxity. Moreover, alterations in both perichondrium and periosteum seems to support our previous hypotheses about the control of long-bone growth exerted by elastic fibers.     

Rabbit Tibial Periosteum and Saphenous Arteriovenous Vascular Bundle as an In Vivo Bioreactor to Construct Vascularized Tissue‐Engineered Bone: A Feasibility Study

The aim of this project was to construct vascularized tissue‐engineered living bone with an autologous vascular network by means of a rabbit bioreactor in vivo. The key components of the in vivo bioreactor for bone formation were the vascularized tibial periosteum and the saphenous vascular bundle. Beta‐tricalcium phosphate (β‐TCP) scaffolds were implanted into the in vivo bioreactor (vascular pedicle implantation and vascularized periosteum encapsulation). At 4 weeks postsurgery, new bone formation was mainly “cartilage‐bone inducing” in the inner periosteum, and was primarily seen in the outer aspects of the scaffold with some amount in the middle part as well. Microvascular infusion showed that direct revascularization of β‐TCP was obtained by means of vascular implantation. Triple staining results showed a large amount of blue collagen fibers. Vascular endothelial growth factor immunohistochemical staining displayed endothelial cells of new blood vessels in bone tissue. The bioreactor established in this study can be used to prepare tissue‐engineered bone with a vascular network.     

Electrospun fibers as a scaffolding platform for bone tissue repair

The purpose of the study is to investigate the effects of electrospun fiber diameter and orientation on differentiation and ECM organization of bone marrow stromal cells (BMSCs), in attempt to provide rationale for fabrication of a periosteum mimetic for bone defect repair. Cellular growth, differentiation, and ECM organization were analyzed on PLGA‐based random and aligned fibers using fluorescent microscopy, gene analyses, electron scanning microscopy (SEM), and multiphoton laser scanning microscopy (MPLSM). BMSCs on aligned fibers had a reduced number of ALP+ colony at Day 10 as compared to the random fibers of the same size. However, the ALP+ area in the aligned fibers increased to a similar level as the random fibers at Day 21 following stimulation with osteogenic media. Compared with the random fibers, BMSCs on the aligned fibers showed a higher expression of OSX and RUNX2. Analyses of ECM on decellularized spun fibers showed highly organized ECM arranged according to the orientation of the spun fibers, with a broad size distribution of collagen fibers in a range of 40–2.4 μm. Taken together, our data support the use of submicron‐sized electrospun fibers for engineering of oriented fibrous tissue mimetic, such as periosteum, for guided bone repair and reconstruction. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1382–1389, 2013     

Lumbar dura mater biomechanics: experimental characterization and scanning electron microscopy observations.

There is no consensus about the anatomical structure of human dura mater. In particular, the orientation of collagen fibers, which are responsible for biomechanical behavior, is still controversial. The aim of this work was to evaluate the mechanical properties and the microstructure of the lumbar dura mater. We performed experimental mechanical characterization in longitudinal and circumferential directions and a scanning electron microscopy observation of the tissue. Specimens of human dura mater were removed from the dorsal-lumbar region (T12-L4/L5) of six subjects at autopsy; specimens of bovine dorsal-lumbar dura mater were obtained from two animals at slaughter. Human and bovine tissues both exhibited stronger tensile strength and stiffness in the longitudinal than in the circumferential direction. Scanning electron microscopy observations of dura mater showed that the collagen fibers are mainly oriented in a longitudinal direction, which accounts for its stronger tensile strength in this direction. We conclude that dura mater has a different mechanical response in the two directions investigated because the fiber orientation is predominantly longitudinal. IMPLICATIONS: In this experimental work, we studied the structural and functional relationship of human lumbar dura mater. We performed mechanical tests and microscopic observations on dura mater samples. The results show that the dura mater is mainly composed of longitudinally oriented collagen fibers, which account for higher tissue resistance in this direction.     

先天性胫骨假关节（CPT）的超微结构

目的：深入了解先天性胫骨假关节（CPT）的病理变化、细菌类型及其病变来源。方法：取5例先天性胫骨假关节（CPT）标本中不同部位的组织20例,经固定、切片染色后置于透射电镜下进行观察。结果：（1）断端间组织及病变骨膜组织性质完全一样,均为致密纤维结缔组织,细胞成分多,主要为纤维母细胞、肌纤维母细胞及少量未分化细胞。（2）移行部位骨膜组织结构和病变处骨膜组织结构相似。（3）断端处骨质细胞稀少,部分骨细胞萎缩或坏死,部分骨细胞含有空泡,骨基质未见异常。（4）移行部位骨质未见明显异常表现。结论：（1）先天性胫骨假关节（CPT）是非神经起源的、而是起源于骨膜的一种细胞增生活跃的纤维增生性变;（2）肌纤维母细胞与CPT发病有关。     

Retinoic acid responsiveness of cells and tissues in developing fetal limbs evaluated in a RAREhsplacZ transgenic mouse model

Limb morphogenesis is a complex phenomenon in which retinoids play an important role. Abnormal maternal retinoid levels from high oral doses cause fetal malformations, including abnormalities of the musculoskeletal system. Our purpose was to identify the retinoid-responsive cells in bone and cartilage during limb development by using a transgenic line of mice containing a reporter gene insert consisting of a retinoic acid response element linked to an Escherichia coli β-galactosidase gene. Transgenic fetuses from day 11.5 after conception to birth (day 20) were analyzed histologically. Retinoid-responsive cells and tissues were first seen in the limb bud at 12.5 days in the webs between the forming digits. The webs stained maximally at 14.5 days, after which staining intensity subsided. Staining in the muscles was detectable at 13.5 days, at a stage coinciding with myoblast fusion. Specific regions of perichondrium and periosteum also stained at this Stage. Occasional staining was observed in individual chondroblasts in all chondrogenic regions, including hypertrophic chondroblasts and certain articular surfaces of developing joints. Staining of these tissues decreased in intensity in subsequent stages. Osteoclasts started to express β-galactosidase at 15.5 days and continued to stain into maturity. Our results indicate that specific subsets of cells respond to retinoids at specific stages in the course of normal limb development. In hypertrophic chondrocytes and cells in the webs and joints that display such a response, retinoid-induced effects may be linked to cell death that occurs in these regions. Staining in muscle, perichondrium, and periosteum may reflect retinoid-induced effects associated with cell differentiation and growth. These results suggest that retinoids play a role in a variety of tissues, including bone and cartilage, at specific stages during morphogenesis.     

Periosteum,bone's “smart” bounding membrane,exhibits direction‐dependent permeability

The periosteum serves as bone's bounding membrane, exhibits hallmarks of semipermeable epithelial barrier membranes, and contains mechanically sensitive progenitor cells capable of generating bone. The current paucity of data regarding the periosteum's permeability and bidirectional transport properties provided the impetus for the current study. In ovine femur and tibia samples, the periosteum's hydraulic permeability coefficient, k, was calculated using Darcy's Law and a custom‐designed permeability tester to apply controlled, volumetric flow of phosphate‐buffered saline through periosteum samples. Based on these data, ovine periosteum demonstrates mechanically responsive and directionally dependent (anisotropic) permeability properties. At baseline flow rates comparable to interstitial fluid flow (0.5 µL/s), permeability is low and does not exhibit anisotropy. In contrast, at high flow rates comparable to those prevailing during traumatic injury, femoral periosteum exhibits an order of magnitude higher permeability compared to baseline flow rates. In addition, at high flow rates permeability exhibits significant directional dependence, with permeability higher in the bone to muscle direction than vice versa. Furthermore, compared to periosteum in which the intrinsic tension (pre‐stress) is maintained, free relaxation of the tibial periosteum after resection significantly increases its permeability in both flow directions. Hence, the structure and mechanical stress state of periosteum influences its role as bone's bounding membrane. During periods of homeostasis, periosteum may serve as a barrier membrane on the outer surface of bone, allowing for equal albeit low quiescent molecular communication between tissue compartments including bone and muscle. In contrast, increases in pressure and baseline flow rates within the periosteum resulting from injury, trauma, and/or disease may result in a significant increase in periosteum permeability and consequently in increased molecular communication between tissue compartments. Elucidation of the periosteum's permeability properties is key to understanding periosteal mechanobiology in bone health and healing, as well as to elucidate periosteum structure and function as a smart biomaterial that allows bidirectional and mechanically responsive fluid transport. © 2013 American Society for Bone and Mineral Research.     

Bone in dermatosparaxis I. Morphologic analysis

Normal (N-) calf bone consists of lamellae regularly spaced and oriented parallel to the periosteum. The lamellae increase in thickness from the periosteum to the medullary cavity, by apposition of layers of cells and a calcifying matrix on either side of a hypercalcified primer. In the dermatosparactic (D-) bone, the hypercalcified primer is barely visible and the cells are irregularly arranged within the lamellae. The poorly defined vascular spaces are partly filled with an acellular calcified material. In the D-bone, the collagen fibers are sparse and radiate from the vascular space, while in the N-bone they are abundant and laid down concentric with the blood channels. In the D-bone, only a few weeks old, the outer lamellae are radially oriented with respect to the medullar cavity, while haversian remodeling already occurs in the inner part of the diaphysis. At 6 months, the inner half of the diaphysis is made up of normal haversian secondary bone, while the outer half is made up of radial lamellae. The alteration of the mechanical properties of procollagen fibers in the D-bone might be responsible for its defective organization. A resistant fibrous framework, therefore, seems required to ensure the spatial organization of the cells in the calcifying matrix and to maintain its cohesion.     

Bone in dermatosparaxis

Normal (N-) calf bone consists of lamellae regularly spaced and oriented parallel to the periosteum. The lamellae increase in thickness from the periosteum to the medullary cavity, by apposition of layers of cells and a calcifying matrix on either side of a hypercalcified primer. In the dermatosparactic (D-) bone, the hypercalcified primer is barely visible and the cells are irregularly arranged within the lamellae. The poorly defined vascular spaces are partly filled with an acellular calcified material. In the D-bone, the collagen fibers are sparse and radiate from the vascular space, while in the N-bone they are abundant and laid down concentric with the blood channels. In the D-bone, only a few weeks old, the outer lamellae are radially oriented with respect to the medullar cavity, while haversian remodeling already occurs in the inner part of the diaphysis. At 6 months, the inner half of the diaphysis is made up of normal haversian secondary bone, while the outer half is made up of radial lamellae. The alteration of the mechanical properties of procollagen fibers in the D-bone might be responsible for its defective organization. A resistant fibrous framework, therefore, seems required to ensure the spatial organization of the cells in the calcifying matrix and to maintain its cohesion.     

Mechanics of Avian Fibrous Periosteum: Tensile and Adhesion Properties During Growth

We report the results of direct mechanical tests of the fibrous periosteum from the tibiotarsi of white leghorn chicks at 4, 6, 8, 9, 10, 11, 12, and 14 weeks of age using a newly developed sample isolation technique. Additionally, this technique allows the determination of the apparent in vivo load on the fibrous periosteum. The periosteum has a highly nonlinear stress-strain relationship at all ages. For loading below the in vivo level, the periosteum is pliant and mean tensile modulus is 3.35 MPa (±1.84 SD, n = 75). For loading above the in vivo level, tensile stiffness is nearly two orders of magnitude greater. In the region of high stiffness, mean modulus is 229.5 MPa (±89.6, n = 72). In vivo, the periosteum is loaded at the transition between these two stiffness regions. We interpret this as indicating that, in vivo, the collagen fibers of the periosteum are aligned, but subject to minimal loading. Stress levels in the periosteum corresponding to in vivo conditions indicate modest loading, and mean apparent in vivo stress levels are 0.92 MPa (±0.37 SD, n = 67). A second technique demonstrated that the adhesion of the periosteum in the diaphyseal region (1–6 weeks of age) is minimal, but is substantial in the metaphyseal region. The metaphyseal adhesion will affect the transmission of load between the physes. These studies suggest that growth of the fibrous periosteum follows the longitudinal growth of the bone, rather than the periosteum having a direct mechanical influence on growth plate activity. Comparison of tensile properties over the course of growth indicates a substantial increase in periosteal stiffness in the early portion of the growth period, which reaches a maximum at approximately 9 weeks posthatching. There is also a marked decline in periosteal stiffness as growth rate declines in the latest stages of growth (14 weeks). This suggests that the basic properties of periosteal collagen may undergo a transition during the course of this tissue’s brief functional lifetime; that is, during long bone growth.     

Osteogenic ability of free periosteal autografts in tibial fractures with severe soft tissue damage: an experimental study

OBJECTIVE: The present study was undertaken to assess whether free nonvascularized autologous periosteum transplants enhance bone healing in a rabbit fracture model designed to resemble a tibial fracture with severe soft tissue damage. DESIGN: Transplantation of free autologous periosteal grafts on the anteromedial site of the tibia (experimental group) was compared with nontransplantation on the contralateral tibia (control group). We produced a standardized transverse osteotomy of both tibial diaphyses in white male adult New Zealand rabbits. The endomedullary cavity was reamed and nailed, and then a one-centimeter segment of periosteum was excised from either side of the osteotomy. To prevent periosteal and extraosseous ingrowth at the osteotomy site, a silastic sheet was wrapped around two-thirds of the circumference of the tibia. In the first group, on the silastic-free bone window, we then spanned the osteotomy with a free, nonvascularized, longitudinally oriented autologous periosteum and sewed it to the adjacent periosteum both proximally and distally. In the second group, the periosteum was placed transversely, leaving a gap between it and the adjacent periosteum proximally and distally. Revascularization of the graft was determined with the colored microsphere technique. MAIN OUTCOME MEASUREMENTS: Histomorphometric analysis of the periosteal callus was done on a transparent grid superimposed on enlarged photographs of the histologic sections. RESULTS: Free, nonvascularized, longitudinally placed autologous periosteum in contact with intact periosteum produced significantly more periosteal callus than was seen in the control group, in which no periosteal graft was used. However, when transversely placed periosteal grafts were set in the silastic-free bone window and there was no contact with surrounding remnants of intact periosteum, no significant difference in callus production was noted when compared with the control. Revascularization of these grafts was seen within one week after transplantation. Bone healing occurred mainly through endochondral ossification. CONCLUSION: Our data suggest that orthotopically placed autologous nonvascularized periosteum retains its osteogenic potential in a poorly vascularized environment such as a tibial fracture with severe soft tissue damage. The effect is enhanced if the graft is in contact with intact periosteum. Histologically, callus formation after periosteal grafting resembles endochondral and intramembranous ossification.     

Cathepsin K deficiency in pycnodysostosis results in accumulation of non-digested phagocytosed collagen in fibroblasts

The rare osteosclerotic disease, pycnodysostosis, is characterized by decreased osteoclastic bone collagen degradation due to the absence of active cathepsin K. Although this enzyme is primarily expressed by osteoclasts, there is increasing evidence that it may also be present in other cells, including fibroblasts. Since fibroblasts are known to degrade collagen intracellularly following phagocytosis, we analyzed various soft connective tissues (periosteum, perichondrium, tendon, and synovial membrane) from a 13-week-old human fetus with pycnodysostosis for changes in this collagen digestion pathway. In addition, the same tissues from cathepsin K-deficient and control mice were analyzed. Microscopic examination of the human fetal tissues showed that cross-banded collagen fibrils had accumulated in lysosomal vacuoles of fibroblasts. Morphometric analysis of periosteal fibroblasts revealed that the volume density of collagen-containing vacuoles was 18 times higher than in fibroblasts of control patients. A similar accumulation was seen in periosteal fibroblasts of three children with pycnodysostosis. In contrast to the findings in humans, an accumulation of internalized collagen was not apparent in fibroblasts of mice with cathepsin K deficiency. Our observations indicate that the intracellular digestion of phagocytosed collagen by fibroblasts is inhibited in humans with pycnodysostosis, but probably not in the mouse model mimicking this disease. The data strongly suggest that cathepsin K is a crucial protease for this process in human fibroblasts. Murine fibroblasts may have other proteolytic activities that are expressed constitutively or up regulated in response to a deficiency of cathepsin K. This may explain why cathepsin K-deficient mice lack the dysostotic features that are prominent in patients with pycnodysostosis.     

Osteochondrogenesis of Free Periosteal Grafts in the Rabbit Iliac Crest

The influence of the bone marrow, cortical bone and apophyseal cartilage of the iliac crest on osteochondrogenesis from free autogenous periosteal grafts was studied histologically in 8-week-old rabbits. Tibial periosteum was transplanted around the iliac crest, from which the periosteum had been removed from the inner side, periosteum and cortical bone in an area on the outer side and perichondrium from the apophyseal cartilage. Most bone formation occurred in the area with periosteum in contact with the bone marrow of the cancellous bone. By means of an isolating Nucleopore filter^®, it was revealed that the most vigorous of this bone formation originated from the periosteal graft. Further, it was noted that in the series with the Nucleopore filter, bone formation was slower than in the series without the filter, suggesting some inductive factors. No bone formation occurred in the apophyseal area.     

Purified collagen I oriented membrane for tendon repair: An ex vivo morphological study

Injured tendons have limited repair ability after full‐thickness lesions. Tendon regeneration properties and adverse reactions were assessed ex vivo in an experimental animal model using a new collagen I membrane. The multilamellar membrane obtained from purified equine Achilles tendon is characterized by oriented collagen I fibers and has been shown to sustain cell growth and orientation in vitro. The central third of the patellar tendon (PT) of 10 New Zealand White rabbits was sectioned and grafted with the collagen membrane; the contralateral PT was cut longitudinally (sham‐operated controls). Animals were euthanized 1 or 6 months after surgery, and tendons were subjected to histological and Synchrotron Radiation‐based Computed Microtomography (SRµCT) examination and 3D structure analysis. Histological and SRµCT findings showed satisfactory graft integration with native tendon. Histological examination also showed ongoing angiogenesis. Adverse side‐effects (inflammation, rejection, calcification) were not observed. The multilamellar collagen I membrane can be considered as an effective tool for tendon defect repair and tendon augmentation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 738–745, 2013     

Histologic anatomy of the triangular fibrocartilage.

The collagen arrangement of the triangular fibrocartilage complex was studied in 20 fresh cadaver wrists by means of standard and polarized light microscopy and scanning electron microscopy. The collagen fibres in the articular disk are arranged in undulating sheets oriented at oblique angles to each other. The fibers of the radioulnar ligaments are oriented longitudinally from the radial origin to the ulnar insertion. The origin of the articular disk from the radius is characterized by thick fibers 1 to 2 mm in length radiating from the radius into the articular disk. Five specimens were also injected with india ink. The radioulnar ligaments and the peripheral 15% to 20% of the articular disk are well vascularized, whereas the central 80% of the articular disk is avascular.     

Radial tie fibers influence the tensile properties of the bovine medial meniscus

Although collagen fibers are arranged predominantly in the circumferential direction in the knee meniscus, there is evidence for radially oriented fibers within human menisci. A bovine medial meniscus model was used to study the hypothesis that radial fibers alter the radial tensile properties of the meniscus. The architecture of the collagen network and tensile properties of the bovine medial meniscus were examined; attention was given to large “radial tie fibers” and their regional variation. Menisci were sectioned serially into slices 400 μm thick. Polarized light microscopy showed that the distribution of radial tie fibers varied greatly among the anterior, central, and posterior regions. These radial tie fibers were larger and more frequent in the posterior region. Radial fibers persisted over many adjacent sections with similar architecture, which led to our hypothesis that they may be arranged in continuous sheets in which the morphology varies by region. Radially oriented specimens for tensile testing were grouped according to the number of radial tie fibers (full, partial, and no fiber) and region (anterior, central, and posterior). Uniaxial tensile testing was performed on a testing machine at a strain rate of 0.00017 sec^?1 until failure. The tensile modulus, ultimate tensile stress, and ultimate tensile strain were determined. The presence of radial tie fibers in the specimen had a significant effect on the tensile modulus and ultimate tensile stress. Specimens containing full radial tie fibers were stiffest and failed at the lowest strains; in specimens from the posterior region, the tensile modulus was 392%, the ultimate tensile stress was 314%, and the ultimate tensile strain was 68% that of the specimens with no radial fibers. In no-fiber specimens, the tensile modulus in the posterior region was 225% of the modulus in the anterior region, and the ultimate tensile strain in the posterior region was 68% that of the strain in the anterior region. The abundance of radial tie fibers in the posterior region seems to contribute to the increased stiffness of this region. The preferential stiffening of the posterior region by these radial fibrous sheets may be well suited to the manner in which the bovine medial meniscus functions in load-bearing.     

Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth

The IGF axis is important for long bone development, homeostasis and disease. The activities of IGF-I and IGF-II are regulated by IGF binding proteins (IGFBPs). IGF-I and IGFBP2 are co-expressed in dynamic fashions in the developing long bones of the chick wing, and we have found that IGF-II is present in the cartilage model and surrounding perichondrium, proliferative and hypertrophic chondrocytes and developing periosteum. To gain insight into endogenous roles of IGF-I, IGF-II and IGFBP2 in long bone development, we have overexpressed IGFBP2 in the developing skeletal elements of the embryonic chick wing in vivo, using an RCAS retroviral vector. IGFBP2 overexpression led to an obvious shortening of the long bones of the wing. We have investigated, at the cellular and molecular levels, the mechanism of action whereby IGFBP2 overexpression impairs long bone development in vivo. At an early stage, IGFBP2 excess dramatically inhibits proliferation by the chondrocytes of the cartilage models that prefigure the developing long bones. Later, IGFBP2 excess also reduces proliferation of the maturing chondrocytes and attenuates proliferation by the perichondrium/developing periosteum. IGFBP2 excess does not affect morphological or molecular indicators of chondrocyte maturation, osteoblast differentiation or cell/matrix turnover, such as expression of Ihh, PTHrP, type X collagen and osteopontin, or distribution and relative abundance of putative clast cells. We also have found that IGFBP2 blocks the ability of IGF-I and IGF-II to promote proliferation and matrix synthesis by wing chondrocytes in vitro. Together, our results suggest that the mechanism of action whereby IGFBP2 excess impairs long bone development is to inhibit IGF-mediated proliferation and matrix synthesis by the cartilage model; reduce the proliferation and progression to hypertrophy by the maturing chondrocytes; and attenuate proliferation and formation of the periosteal bony collar. These actions retard the growth and longitudinal expansion of the developing long bones, resulting in shortened wing skeletal elements. Our results emphasize the importance of a balance of IGF/IGFBP2 action at several stages during normal long bone development.     

Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth

The IGF axis is important for long bone development, homeostasis and disease. The activities of IGF-I and IGF-II are regulated by IGF binding proteins (IGFBPs). IGF-I and IGFBP2 are co-expressed in dynamic fashions in the developing long bones of the chick wing, and we have found that IGF-II is present in the cartilage model and surrounding perichondrium, proliferative and hypertrophic chondrocytes and developing periosteum. To gain insight into endogenous roles of IGF-I, IGF-II and IGFBP2 in long bone development, we have overexpressed IGFBP2 in the developing skeletal elements of the embryonic chick wing in vivo, using an RCAS retroviral vector. IGFBP2 overexpression led to an obvious shortening of the long bones of the wing. We have investigated, at the cellular and molecular levels, the mechanism of action whereby IGFBP2 overexpression impairs long bone development in vivo. At an early stage, IGFBP2 excess dramatically inhibits proliferation by the chondrocytes of the cartilage models that prefigure the developing long bones. Later, IGFBP2 excess also reduces proliferation of the maturing chondrocytes and attenuates proliferation by the perichondrium/developing periosteum. IGFBP2 excess does not affect morphological or molecular indicators of chondrocyte maturation, osteoblast differentiation or cell/matrix turnover, such as expression of Ihh, PTHrP, type X collagen and osteopontin, or distribution and relative abundance of putative clast cells. We also have found that IGFBP2 blocks the ability of IGF-I and IGF-II to promote proliferation and matrix synthesis by wing chondrocytes in vitro. Together, our results suggest that the mechanism of action whereby IGFBP2 excess impairs long bone development is to inhibit IGF-mediated proliferation and matrix synthesis by the cartilage model; reduce the proliferation and progression to hypertrophy by the maturing chondrocytes; and attenuate proliferation and formation of the periosteal bony collar. These actions retard the growth and longitudinal expansion of the developing long bones, resulting in shortened wing skeletal elements. Our results emphasize the importance of a balance of IGF/IGFBP2 action at several stages during normal long bone development.