Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates.

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.     

Evaluation of Activity of Epiphyseal Plates in Growing Males and Females

To investigate the age-related activity of the epiphyseal plates, a retrospective study of ^99mTc-methylene diphosphonate bone scans was undertaken. The study comprised 81 males and 46 females aged 2 weeks to 24 years. The total percentage (%) whole-body (ratio of total physis activity to whole-body activity) and the regional % whole-body (ratio of physis activity of one region to whole-body activity) were derived. The ratio of physis activity of one region to the total physis activity was defined as % physis. Before age 12, total physis activity was found to contribute about 10% to whole-body activity. All total and regional % whole-body activities followed sigmoid curves with age. The differences of the parameters (transition centers and widths) suggested that there might be a later and longer period for the disappearance of physis activity in males than in females. For all the regions, % physis changed little with age until after puberty. At age <1, the proportion of bone activity in the body was about 30–35% for skull, 20–25% for lower limbs, and 5–15% for the rest of the regions. The maximal changes during growth occurred in the skull and the lower limbs. The age-related changes of physis activity during growth reflect a combination of the potential of bone to grow and the processes of bone growth and bone turnover. Bone scintigraphy is useful in understanding the changes of physis activity during growth.     

Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.     

Bone lengthening osteogenesis,a combination of intramembranous and endochondral ossification: an experimental study in sheep

We evaluated the morphological features of the newly formed tissue in an experimental model of tibial callotasis lengthening on 24 lambs, aged from 2 to 3 months at the time of operation. A unilateral external fixator prototype Monotube Triax^® (Stryker Howmedica Osteonics, New Jersey) was applied to the left tibia. A percutaneous osteotomy was performed in a minimally traumatic manner using a chisel. Lengthening was started 7 days after surgery and was continued to 30 mm. The 24 animals were randomly divided into three groups of 8 animals each: in Group 1, lengthening took place at a rate of 1 mm/day for 30 days; in Group 2, at a rate of 2 mm/day for 15 days; in Group 3, at a rate of 3 mm/day for 10 days. In each group, 4 animals were killed 2 weeks after end of lengthening, and the other 4 animals at 4 weeks after end of lengthening. To assess bony formation in the distraction area, radiographs were taken every 2 weeks from the day of surgery. To study the process of vascularization, we used Spalteholz’s technique. After killing, the tibia of each animal was harvested, and sections were stained with hematoxylin and eosin, Masson’s trichrome, and Safranin-O. Immunohistochemistry was performed, using specific antibodies to detect collagens I and II, S100 protein, and fibronectin. A combination of intramembranous and endochondral ossification occurred together at the site of distraction. Our study provides a detailed structural characterization of the newly formed tissue in an experimental model of tibial lengthening in sheep and may be useful for further investigations on callotasis.     

Lectin-binding histochemistry of intracellular and extracellular glycoconjugates of the reserve cell zone of growth plate cartilage

The distribution of intracellular and extracellular lectin-binding glycoconjugates of the reserve cell zone of growth plate cartilage was studied in the distal radial growth plate of 4-week-old Yucatan swine using a postembedment method on Epon-embedded sections. Direct comparisons were made to articular, tracheal, and auricular cartilages not involved in endochondral ossification. All patterns of lectin binding that in the growth plate were restricted to the reserve cell zone were also patterns characteristic of tracheal, articular, and auricular cartilages. These included: (a) pericellular binding with peanut agglutinin (PNA) without prior digestion with neuraminidase; (b) pericellular binding with wheat germ agglutinin (WGA) at 24 h; (c) intracellular cytoplasmic binding to concanavalin A (CON-A), Lens culinaris agglutinin (LCA), and Lotus tetragonobolus agglutinin (LTA) after periodic acid oxidation; and (d) a lack of pericellular binding with CON-A and ricin agglutinin 1 (RCA-1) after periodic acid oxidation. We conclude that reserve zone chondrocytes lack specific phenotypic markers as defined by lectin-binding affinity that are found in the cellular zones of the growth plate that undergo calcification and vascularization. The reserve zone has identical lectin-binding affinities to the three structural cartilages used as controls. One interpretation of these results is that the reserve zone may not be involved directly in endochondral ossification, but may have a structural function in growth plate cartilage.     

Changes in osteonectin distribution and levels are associated with mineralization of the chicken tibial growth cartilage

Summary Osteonectin is a calcium-binding matrix protein thought to play a role in regulating calcium distribution in a variety of biologic processes. To examine its role in endochondral bone formation, we examined the distribution of the protein during mineralization of the chicken tibial growth cartilage, using immunohistochemistry and immunoelectron microscopy. The synthesis of osteonectin was also determined in chondrocyte populations isolated from premineralizing and mineralizing regions of growth cartilage and assayed in short-term culture. The resuls show that a very low level of osteonectin is detectable in the resting, proliferating, and early hypertrophic zones of growth cartilage; in these zones, osteonectin is largely cell-associated. In contrast, a large amount of osteonectin is present in the mineralizing zone where it is associated with the matrix. Biosynthetic data from short-term culture experiments indicate, however, that osteonectin is synthesized and secreted by chondrocytes from both premineralizing and mineralizing zones. As indicated by immunoprecipitation, Northern hybridization,in vitro translation of hybrid-selected messenger RNA (mRNA), and electrophoretic analysis, osteonectin synthesized by chondrocytes of the premineralizing zones is not obviously different in structure from that synthesized by chondrocytes of the mineralizing zone. We conclude that osteonectin is a product of chondrocytes in each zone of growth cartilage but accumulates only in the mineralizing zone. The high affinity of the protein for calcium could favor its retention in calcifying matrix.     

Enhancement of Crude Bone Morphogenetic Protein-Induced New Bone Formation and Normalization of Endochondral Ossification by Bisphosphonate Treatment in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. We investigated both the quantity and quality of heterotopic new bone induced by crude bone morphogenetic protein (BMP) as a means of examining bone metabolism by bisphosphonate administration in OPG^−/− mice. Four weeks after implantation of crude BMP, the volume of heterotopic new bone in OPG^−/− mice without alendronate was significantly less than in wild-type (WT) mice. Alendronate treatment of OPG^−/− mice resulted in enhancement of the volume of heterotopic new bone. Histological findings revealed that WT mice showed normal bone formation with persistent cartilage that was interspersed with islands of bone. In contrast, the cartilage was replaced by trabecular bone and bone marrow adipocytes in OPG^−/− mice without alendronate. However, some cartilage was still present in OPG^−/− mice with alendronate compared to those without alendronate. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG^−/− mice with alendronate than in those without alendronate. These findings suggest that in stimulated osteoclastogenesis without OPG, osteoinductive activity induced by crude BMP is inhibited and endochondral ossification induced by crude BMP is accelerated. On the other hand, alendronate treatment of OPG^−/− mice caused osteoinductive activity induced by crude BMP to increase and endochondral ossification induced by crude BMP to be decelerated. In conclusion, inhibition of stimulated osteoclastogenesis results in the enhancement of new bone formation and normalization of endochondral ossification.     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

松质骨骨基质明胶负载软骨细胞体外培养的实验研究

目的　探索松质骨骨基质明胶 (BMG)作为软骨细胞培养支架的可行性。方法　分离幼兔关节软骨细胞 ,体外单层培养扩增 ,并负载于松质骨BMG上体外培养 ,于不同时间取材进行解剖显微镜、组织学、透射电镜观察及免疫组化检测。结果　培养 6d软骨细胞在松质骨BMG上分裂增殖。 1 2d形成 1 0～ 1 2层细胞的软骨组织 ,Safranin -O染色阳性。 1 8d软骨组织厚度增加 ,胶原染色阳性。培养 2 4～ 42d网眼中细胞数量增多 ,周围形成 2 5～ 30层细胞的软骨组织。培养 42d ,解剖显微镜观察显示形成直径 4mm的“圆盘状软骨”。电镜观察显示软骨细胞外有胶原纤维。免疫组化检测显示形成的软骨组织基质中有Ⅱ型胶原。结论　松质骨BMG可作为软骨细胞培养的较好支架     

微小RNAs在骨与软骨组织中的调节作用

目的对微小RNAs(microRNAs,miRNAs)在骨与软骨组织中的调控作用和作用机制作一综述。方法广泛查阅近年来有关miRNAs在骨与软骨组织的调控作用及作用机制的文献,进行总结与分析。结果 miRNAs是近年来骨关节疾病研究的热点,越来越多研究显示其在骨与软骨组织形成和代谢过程中,对于细胞分化、细胞基质分泌等方面发挥重要的调控作用,但确切机制尚不清楚。结论 通过对miRNAs在骨与软骨组织中的调控作用及作用机制的研究,可能为了解退行性骨关节疾病开辟新的领域。     

Reimplantation of growth plate chondrocytes into growth plate defects in sheep

Defects in growth plates due to trauma, infection, or genetic causes can result in bone formation across the defect, bridging the epiphysis and metaphysis, resulting in growth arrest and limb deformation. We have investigated the capacity of implanted chondrocyte cultures to prevent this process. Sheep growth plate chondrocytes were isolated, and after culture at high density produced easily manipulated cartilaginous discs. The tissue was implanted into growth plate defects produced in lambs and the response was assessed histologically. Following implantation, cultures continued to proliferate and maintain a cartilage-like matrix. After 8 to 12 weeks, hypertrophic maturation chondrocyte columnation, and associated endochondral calcification were observed. Culture implantation was always associated with local immune inflammatory reaction, which continued throughout the course of investigation. Cellular survival was variable and resulted in the presence of viable implants as well as residual cartilage matrix devoid of chondrocytes; however, implanted chondrocyte discs always prevented bone bridge formation. These findings encourage the expectation that cultured chondrocytes may provide a useful replacement for the inert interpositional materials currently used in the treatment of growth arrest. The potential of this technique for growth plate replacement, however, requires a more predictable rate of implant survival. The likely reasons for implant loss are discussed.     

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 × 10⁶ cells per 35-mm plate and grown for 17 days in BGJ_b medium supplemented with 10% fetal bovine serum, 50 μg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive ⁴⁰Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.     

Extracellular matrix alterations during endochondral ossification in humans

Immunohistochemical methods were employed to examine alterations in the cartilage extracellular matrix constituents associated with endochondral ossification in humans. The distributions of chondroitin 4- and 6-sulfate and keratan sulfate proteoglycan (PG) determinants, cartilage PG link protein, collagen types I and II, and fibronectin were determined in iliac crest growth-plate specimens using the avidin-biotin-horseradish peroxidase system. Collagen type II was distributed throughout the growth plate, providing a framework within which chondrocytes divided and formed clusters of differentiating (hypertrophic) cells. The septa between these clusters and their subchondral extensions into underlying bone trabeculae were rich in PG, PG link protein, and collagen type II and resembled the extracellular matrix of reserve cartilage. The territorial matrix associated with the differentiating cells within the clusters contained reduced amounts of collagen type II, PG link protein, and possibly cartilage PG. Collagen type I and fibronectin were detected within the cytoplasm of the maturing and degenerating cells, and fibronectin localized intensely to the pericellular matrix envelopes of these cells. These alterations presumably facilitate the degradation of the matrix associated with the cell clusters by invading vascular tissue, while the septa, which retain the characteristics of more typical cartilage matrix, are not degraded and firmly anchor the cartilage to the subchondral bone.     

P-glycoprotein is expressed in the mineralizing regions of the skeleton

Using oligonucleotide primers specific for the human MDR 1 gene, we were able to identify a specific amplicon using RT-PCR from total bovine growth plate chondrocyte RNA. The identification of MDR mRNA in growth plate chondrocytes led us to examine the precise distribution of MDR P-glycoprotein in bone and cartilage. We applied two monoclonal antibodies (C219 and C494) to human fetal, neonatal, and childhood growth plates and bone. In growth plates, P-glycoprotein was detected at high levels in a perilacunar distribution in the calcifying zone and at lower levels in hypertrophic, but not proliferative or reserve zone, chondrocytes. P-glycoprotein was also observed in perichondrial chondrocytes, in perivascular chondrocytes and matrix in the fetal cartilage anlage, and in osteoblasts and the surface osteoid matrix of newly formed bone trabeculae in the primary spongiosa. The recently described chloride channel of P-glycoprotein suggests a potential role of P-glycoprotein in growth plate chondrocyte hypertrophy. D. C. Mangham is supported by the Wechsler fellowship     

The matrix of endochondral bone differs from the matrix of intramembranous bone

Summary Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induced cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.     

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing.

Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data.

In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group.

In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.     

The role of mechanical loading histories in the development of diarthrodial joints

The possible role of mechanical loading history in chondroosseous development at the ends of long bones is explored using two-dimensional finite element models of chondroepiphyses. Loading histories are characterized in terms of discrete loading cases defined by joint contact pressure distributions and an associated number of loading cycles. An osteogenic stimulus throughout the chondroepiphyses is calculated following the theory that cyclic octahedral shear stresses promote endochondral ossification and cyclic compressive dilatational stresses inhibit ossification. The resulting distributions for the osteogenic stimulus predict the appearance of the secondary ossific nucleus and the shape of the developing bony epiphysis. The zone of Ranvier and the formation of articular cartilage and the growth plate are also predicted by the models. These findings are consistent with the hypothesis that tissue stress histories constitute an important influence during skeletal morphogenesis. Further study and testing of the concepts introduced in this study are appropriate.     

Changes in lipids during matrix: Induced endochondral bone formation

Summary The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (day 11–13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in thein vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7–9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.