Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

Runx1 and Runx2 cooperate during sternal morphogenesis.

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1(f/f) mice and alpha1(II) Runx1(f/f) mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and alpha1(II) DKO mice, respectively]. Prx1 Runx1(f/f) mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, alpha1(II) Runx1(f/f) mice and alpha1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of alpha1(II) collagen (Col2a1 - Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of alpha1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

The essential requirement for Runx1 in the development of the sternum.

Runx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1(Re/Re) embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1(Re/Re) embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements.

Transgenic mice expressing D469Delta mutated cartilage oligomeric matrix protein (COMP) show growth plate abnormalities and sternal malformations.

In humans, mutations in cartilage oligomeric matrix protein (COMP) cause autosomal dominantly inherited skeletal dysplasias. We have generated transgenic mouse lines to study the role of mutant D469Delta COMP in the pathogenesis of pseudoachondroplasia. Biochemical characterization of cartilage tissue demonstrated that transgenic and endogenous COMP subunits were able to form mixed, pentameric molecules in vivo. Mutant COMP was more difficult to extract than the wildtype protein, suggesting an altered anchorage within the matrix. Although both transgenic wildtype and mutant COMP were detected throughout the growth plate, mutant molecules were restricted to the pericellular matrix while wildtype COMP showed a uniform distribution throughout the extracellular matrix. Mice expressing the mutant transgene showed a slight gender specific growth retardation. In mutant animals, the columnar organization in the growth plate was disturbed, proteoglycans were lost and improperly formed collagen fibrils were observed. In some chondrocytes the endoplasmic reticulum was dilated, most probably due to an impaired secretion of mutant COMP similar to that observed in patients. Later in development, the growth plate was irregularly shaped and prematurely invaded by bony tissue. In addition, a fusion of the third and fourth sternebrae was frequently observed.

Connexin 40, a target of transcription factor Tbx5, patterns wrist, digits, and sternum.

Haploinsufficiency of T-box transcription factor 5 (TBX5) causes human Holt-Oram syndrome (HOS), a developmental disorder characterized by skeletal and heart malformations. Mice carrying a Tbx5 null allele (Tbx5(+/Delta)) have malformations in digits, wrists, and sternum joints, regions where Tbx5 is expressed. We demonstrate that mice deficient in connexin 40 (Cx40), a Tbx5-regulated gap junction component, shared axial and appendicular skeletal malformations with Tbx5(+/Delta) mice. Although no role in skeleton patterning has been described for gap junctions, we demonstrate here that Cx40 is involved in formation of specific joints, as well as bone shape. Even a 50% reduction in either Tbx5 or Cx40 produces bone abnormalities, demonstrating their crucial control over skeletal development. Further, we demonstrate that Tbx5 exerts in part its key regulatory role in bone growth and maturation by controlling via Cx40 the expression of Sox9 (a transcription factor essential for chondrogenesis and skeleton growth). Our study strongly suggests that Cx40 deficiency accounts for many skeletal malformations in HOS and that Tbx5 regulation of Cx40 plays a critical role in the exquisite developmental patterning of the forelimbs and sternum.

Enhanced Sternal Healing Through Platelet-Rich Plasma and Biodegradable Gelatin Hydrogel.

Platelet-rich plasma (PRP) contains numerous growth factors and promotes bone fracture healing. The aim of this study was to evaluate the effectiveness of the controlled release of PRP from biodegradable gelatin hydrogel for promoting healing in a rabbit ischemic sternal model. PRP was prepared from the whole blood of a Japanese white rabbit. Sixteen rabbits were randomized into four groups (each n = 4) and all underwent median sternotomy and bilateral internal thoracic artery removal. Before the sternum was closed, the following solutions were applied between the sternum incisions in three of the groups: 30 mg of gelatin hydrogel incorporating 300 µL of phosphate-buffered saline, 300 µL of a solution form of PRP, or 30 mg of gelatin hydrogel incorporating 300 µL of PRP (PRP + Gel). The fourth group acted as a control. Sternal healing was evaluated by histology and microcomputed tomography 7 days after the intervention. The PRP + Gel group showed a significantly higher proportion of fibrosis within the fracture area (an indicator of sternal healing) than the other groups and a significantly higher mean intensity of osteocalcin. These results indicate that the controlled release of PRP from locally applied gelatin hydrogel was markedly effective in enhancing sternal healing in the early postoperative period. This novel therapy could potentially help prevent complications, such as deep sternal wound infection and could result in early postoperative ambulation after median sternotomy.

Decreased Tc-99m sestamibi uptake in a sternal brown tumor indicating response to anti metabolic therapy for secondary hyperparathyroidism.

Brown tumors are rare but serious complications of renal osteodystrophy, and can be treated by parathyroidectomy or by pharmacological treatment of hyperparathyroidism. In addition to parathyroid lesions such as adenoma, hyperplasia, and carcinoma, brown tumors have been detected effectively by using dual phase Tc-99m sestamibi and Tl-201 chloride. We describe an unusual case of brown tumor at the manibrium sterni which shows marked increased Tc-99m sestamibi uptake on the initial scan, with decreasing tracer activity on follow-up scan indicating a response to antimetabolic therapy.

Penetration of moxifloxacin into sternal bone of patients undergoing routine cardiopulmonary bypass surgery.

This study investigated plasma and bone concentrations of moxifloxacin following a single intravenous dose of 400mg to consider its potential role in the treatment of osteomyelitis. Eight patients who underwent routine cardiopulmonary bypass surgery were enrolled in the study. Plasma and bone samples were collected 2h and 5h after the end of infusion. High performance liquid chromatography was used for the determination of moxifloxacin concentrations. Mean plasma concentrations were 3.36 microg/mL and 2.93 microg/mL at 2h and 5h after the end of infusion. The concentrations in the body and manubrium of the sternal bone were 1.65 microg/g and 1.64 microg/g at 2h and 1.4 microg/g and 1.45 microg/g at 5h, respectively. Moxifloxacin showed good penetration into bone and could be considered for the treatment of osteomyelitis.

Radiotoxicity of the alpha-emitting bone-seeker 223Ra injected intravenously into mice: histology, clinical chemistry and hematology.

BACKGROUND: The alpha-emitter 223Ra, which localizes in osteoblastic active zones, including on skeletal surfaces and in osteoblastic metastases, has recently been introduced as a potential therapeutic agent against skeletal metastases. Here, the adverse effects of high dosages in animals were investigated. MATERIALS AND METHODS: Balb/c mice received intravenously (i.v.) either 1250, 2500, or 3750 kBq/kg of dissolved 223RaCl2 and were followed in the initial toxicity phase. At the 4-week end-point, the animals were sacrificed and blood samples were collected to study the effects on clinical chemistry and hematological parameters. Selected organs were weighed and tissue samples examined by microscopy. RESULTS: Treatment with 223Ra caused a dose-related minimal to moderate depletion of osteocytes and osteoblasts in the bones. Furthermore, a dose-related minimal to marked depletion of the hematopoietic cells in the bone marrow, and a minimal to slight extramedullary hematopoiesis in the spleen and in the mandibular and mesenteric lymph nodes were observed. The LD50 for acute toxicity, defined as death within 4 weeks of receiving the substance, was not reached. CONCLUSION: This study demonstrated that high doses of the bone-seeker 223Ra did not completely inactivate the blood-producing cells. The relatively high tolerance to skeletal alpha doses was probably caused by the surviving pockets of red bone marrow cells beyond the range of alpha particles from the bone surfaces, and the recruitment of peripheral stems cells.

Age-related normal variants of sternal uptake on bone scintigraphy.

PURPOSE: The purpose of this study was to describe the variable age-related normal appearance of the sternal area on bone scintigraphy. METHODS: We studied 334 patients (158 males and 176 females) aged 15 days to 85 years, mean age 32.5 +/- 19.9 years, who did not have symptoms associated with the sternum and chest wall or a history of malignancy. All patients underwent whole-body bone scan 3 hours after intravenous injection of Tc-99m MDP. RESULTS: In patients younger than 10 years, sternal uptake was equal to nearby ribs with homogeneous uptake and poorly defined outlines. In older patients, the sternum showed markedly greater uptake than nearby ribs and the borders became well defined. Different patterns of sternal uptake were noted: 1) increased uptake in the manubrium; 2) hot spots in the angle of Louis, body and xiphoid; 3) a photopenic area just above the xiphoid process; 4) heterogeneous sternal body uptake; and 5) hyperactivity along the sternal body borders. Of these variants, the angle of Louis hot spot was the most common finding (63.5%). A linear pattern of hot spots in the sternal body was seen most frequently between 10 and 30 years old and corresponded to 3 transverse ridges of bone, representing the sites of union. Increased tracer uptake only at the medial end of the clavicle in the SC joints was seen in 42.13% of patients younger than 30 years. CONCLUSION: This study showed age-related normal variants of sternal uptake on bone scanning in asymptomatic patients. Understanding these variants may be helpful in differentiating normal and abnormal patterns of uptake in the sternum on radionuclide bone imaging.

The Distribution of Elements in 48 Canine Compact Bone Types Using Handheld X-Ray Fluorescence.

A major question when we talk about the elements in the bone is whether all bones contain the same elements. To answer this question, this study was designed for determination of the elemental levels in 48 various canine compact bones using handheld X-ray fluorescence technique. From a total of 26 elements that could be detected, only 13 elements were found in all 48 bones. The sternum and os penis were significantly different from the other bones in that they contained the highest number of elements. The ratio of Ca and P was significantly different when comparing certain bones: there was a higher Ca/P ratio in the patella (right), calcaneus (right and left), and sternum compared with a lower ratio in the radius (left), rib (left), phalanx (left forelimb), and carpus (left). These results are the first to demonstrate that different types of bones have different elemental profiles, even for major elements such as Ca and P. Moreover, the Ca/P ratio was also different between bone types. This data is important for the selection of bones appropriate to the element studied. In addition, the results proved that the elements were not equally distributed in every bone in the body.

Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis.

Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1-Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes fit a model of cell intercalation along differential Dchs1-Fat4 activity that drives a simultaneous narrowing, thickening and elongation of the sternum. Our results identify the regulation of cellular polarity within the early pre-chondrogenic mesenchyme, when skeletal shape is established, and provide the first demonstration that Fat4 and Dchs1 establish polarized cell behaviour intrinsically within the mesenchyme. Our data also reveal the first indication that cell intercalation processes occur during ventral body wall elongation and closure.

In vivo assessment of bone marrow toxicity by gold nanoparticle-based bioconjugates in Crl:CD1(ICR) mice.

INTRODUCTION: The present study aimed at evaluating the biodistribution of Tween(®) 20-gold nanoparticle (GNP) conjugates and their potential toxicity on the bone marrow before moving on to Phase I clinical trials. MATERIALS AND METHODS: Tween(®) 20-conjugated GNPs were injected intravenously for 21 days in male Crl:CD1(ICR) mice. Body weight of the mice was evaluated each day. After the sub-chronic Tween(®) 20-GNPs administration, blood samples were harvested, and a full blood count was done individually. Total Au quantity from all major organs was assessed using inductively coupled plasma mass spectrometry. One femur and the sternum obtained from each animal were used for histological assessment. RESULTS: Our data showed that the Tween(®) 20-GNP conjugates were found in large quantities in the bladder. Au was shown to accumulate in the hematopoietic bone tissue, with significant side effects such as leucopoiesis and megakaryopoiesis. The mice had a higher white blood cell and platelet count as opposed to the control group. This suggested that the previously described leukopenic effects of isoflurane were overridden by the leucopoietic effects of Tween(®) 20-GNPs. CONCLUSION: It was uncertain whether the mice were reactive to Au as it is a foreign substance to the tissues or whether the side effects observed were a precursor condition of a more severe hematological condition. Au was found to be hepatotoxic, urging the need for further studies in order to achieve better in vivo compliance and exploit the immense potential of GNPs in cancer pharmacology.

T-2 Toxin Alters the Levels of Collagen II and Its Regulatory Enzymes MMPs/TIMP-1 in a Low-Selenium Rat Model of Kashin-Beck Disease.

The objectives of this study are to assess T-2 toxin's involvement in low selenium (Se)-induced Kashin-Beck disease (KBD) in rats and unveil the mechanisms underlying this disease. Two hundred thirty rats were randomly divided into two groups after weaning and fed normal or low-Se diets (n = 115), respectively, for a month. After low-Se model confirmation, rats in each group were subdivided into five: two subgroups (n = 20) were fed their current diets (normal or low-Se diets, respectively) for 30 and 90 days, respectively; two other subgroups (n = 25) received their current diets + low T-2 toxin (100 ng/g BW/day) for 30 and 90 days, respectively; and 25 rats were fed their current diets + high T-2 toxin (200 ng/g BW/day) for 30 days. Articular cartilage samples were extracted for hematoxylin and eosin (H&E) staining and immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to assess protein and mRNA levels, respectively, of collagen II, matrix metalloproteinase (MMP-1), MMP -3, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). Low Se and T-2 toxin synergistically affected animal fitness. Interestingly, low Se + T-2 toxin groups showed KBD characteristics. MMP-1, -3, and -13 mRNA and protein levels generally increased in low-Se groups, while collagen II and TIMP-1 levels showed a downward trend, compared with normal diet fed animals for the same treatment (P < 0.05). T-2 toxin's effect was dose but not time dependent. Low Se and T-2 toxin synergistically alter the expression levels of collagen II as well as its regulatory enzymes MMP-1, MMP-3, MMP-13, and TIMP-1, inducing cartilage damage. Therefore, T-2 toxin may cause KBD in low-Se conditions.

Sternal uptake on bone scintigraphy: age-related variants.

BACKGROUND: When reporting bone scans, it is important to distinguish between normal variants and skeletal pathology involving the sternum. There are only limited reports dealing with age-related normal variants of the sternum on bone scintigraphy. METHODS: We have studied the age-related variants of sternal uptake on bone scintigraphy. In a prospective study, 152 consecutive patients (66 males and 86 females) undergoing whole-body bone scanning, and who had no symptoms associated with the sternum, were evaluated for patterns of normal sternal uptake. Three hours after intravenous injection of (99m)Tc-methylene diphosphonate ((99m)Tc-MDP), whole-body bone scans in the anterior and posterior projections were acquired. Patterns of sternal uptake, including the sites and distribution of increased and decreased uptake, were analysed using age-related groups. RESULTS: Three patterns of tracer uptake in the sternum were recognized: a uniform pattern was most common in children (< or =12 years); a heterogeneous uptake pattern was frequently seen in adolescents, young adult and adult groups; and a segmented pattern was commonly seen in the geriatric group (>60 years). A predominant focal finding was a hot spot at the angle of Louis. In addition, there were focal spots of decreased tracer uptake in the lower sternum, just above the xiphoid process, and spots of increased tracer uptake in the body of the sternum. Such focal spots were not seen in subjects of less than 12 years of age. CONCLUSION: Evolutionary changes of the sternum appear to exist throughout life. There are age-related normal variants of sternal uptake on bone scintigraphy.

Lead concentrations in cortical and trabecular bones in deceased smelter workers.

The aim of the study was to compare bone lead concentrations in cortical and trabecular bones in long-term exposed primary copper and lead smelter workers, and to relate the measured concentrations to the previous lead exposure of the workers. Lead concentrations in seven bones (trabecular: sternum, vertebrae, iliac crest, rib; cortical: femur, left forefinger, and temporal bone) were determined by electrothermal atomic absorption spectrometry in 32 male, long-term exposed copper and lead smelter workers, and compared with levels in 10 male occupationally unexposed reference persons. A time-integrated blood lead index (cumulative blood lead index, CBLI) was calculated for each worker. The lead levels in the seven studied bones were all significantly higher in active and retired lead workers as compared with the reference group (p

Articular chondrocytes produce factors that inhibit maturation of sternal chondrocytes in serum-free agarose cultures: a TGF-beta independent process.

Under normal conditions, articular chondrocytes persist throughout postnatal life, whereas "transient" chondrocytes, which constitute the bulk of prenatal and early postnatal cartilaginous skeleton, undergo maturation, hypertrophy, and replacement by bone cells. The mechanisms regulating the markedly different behavior and fate of articular and transient chondrocytes are largely unclear. In the present study, we asked whether articular chondrocytes possess dominant antimaturation properties which may subtend their ability to persist throughout life. Adult chicken articular chondrocytes and transient maturing chondrocytes from the core region of day 17, chick embryo cephalic sternum were cultured or cocultured in serum-free agarose conditions. When the sternal cells were grown by themselves, they quickly developed into hypertrophic type X collagen-synthesizing cells; however, when they were cocultured with as few as 10% articular chondrocytes or fed with articular chondrocyte-conditioned medium, their maturation was markedly impaired, as revealed by a sharp drop in type X collagen synthesis. A similar, albeit less potent, antimaturation activity characterized resting and proliferating immature chondrocytes isolated from other regions of embryonic sternum. Transforming growth factor-beta 2 (TGF-beta 2) was previously suggested to be an inhibitor of chondrocyte maturation. We found, however, that treatment with a neutralizing antiserum to TGF-beta did not counteract the inhibition of maturation in cocultures of articular and maturing core sternal chondrocytes. Indeed, articular chondrocytes produced and accumulated relatively low levels of TGF-beta in their culture medium, about 15 ng/ml/48 h, of which over 90% was latent; surprisingly, maturing sternal core chondrocytes accumulated over 10-fold more TGF-beta in the medium, about 150 ng/ml/48 h, of which over 20% was endogenously active. These results indicate that articular chondrocytes do possess dominant antimaturation properties which appear to be TGF-beta independent. The TGF-beta s may thus have a more prominent role in the terminal phases of chondrocyte maturation, as indicated by their abundance and greater activity in hypertrophic chondrocytes.

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.