Bony Matrix Research Articles

Evaluation of Potential Roles of Zinc Finger Homeobox 3 (Zfhx3) Expressed in Chondrocytes and Osteoblasts on Skeletal Growth in Mice.

Bone formation is tightly modulated by genetically encoded molecular proteins that interact to regulate cellular differentiation and secretion of bony matrix. Many transcription factors are known to coordinate these events by controlling gene transcription within networks. However, not all factors involved are known. Here, we identified a novel function forZinc Finger Homeobox 3(Zfhx3), a gene encoding a transcription factor, as a regulator of bone metabolism. We knocked outZfhx3conditionally in mice in either chondrocytes or osteoblasts and characterized their bones by micro-CT in 12-week-old mice. We observed a negative effect in linear bone growth in both knockout mice but reduced bone mass only in mice withZfhx3deleted in osteoblasts. Loss of Zfhx3 expression in osteoblasts affected trabecular bone mass in femurs and vertebrae in both sexes but influenced cortical bone volume fraction only in females. Moreover, transcriptional analysis of femoral bones in osteoblastZfhx3conditional knockout mice revealed a reduced expression of osteoblast genes, and histological evaluation of trabecular bones suggests that Zfhx3 causes changes in bone formation and not resorption. The loss of Zfhx3 causes reductions in trabecular bone area and osteoid volume, but no changes in the expression of osteoclast differentiation markers or number of TRAP stained osteoclasts. These studies introduce Zfhx3 as a relevant factor toward understanding gene regulatory networks that control bone formation and development of peak bone mass.

Facile design of biofunctionalized nanocomposite hydrogel to potentiate angiogenesis and osteogenesis for the skull regeneration

The clinical treatment of cranial defect reconstruction using hydrogels faces challenges such as inadequate biomechanical strength and limited biofunctional effects. In this study, we have addressed these issues by developing a novel hydrogel. This hydrogel composes desferrioxamine-modified laponite nanoplatelets (LAP/DFO) combined with tannin-modified poly(vinyl alcohol) (PVA/TA), aiming to closely emulate the natural organic-inorganic bony matrix. Our results indicated that the multifunctional hydrogel system, particularly when incorporating LAP/DFO (referred to as PL10), could form a highly ordered porous structure, achieve appropriate biomechanical strength, and release bioactive factors as expected. This system enhanced the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) for angiogenesis and promoted mesenchymal stem cells (MSCs) osteogenic differentiation for osteogenesis in vitro. Moreover, in vivo investigations confirmed the efficacy of the multifunctional hydrogels, particularly PL10, in enhancing bone regeneration compared to blank PVA. Collectively, this study contributes valuable insights into the design of bioactive factor delivery systems and offers efficient therapeutic strategies for promoting the repair of cranial defects.

Anaesthetic concerns of a child with symptomatic vitamin D deficiency rickets with secondary hyperparathyroidism: A case report.

Rickets is a paediatric bone disorder characterised by defective mineralisation of bony matrix due to abnormalities in calcium and phosphate metabolism. Despite being a common disease globally, literature on the anaesthetic concerns in rickets are scant. Herein, we describe the management of a 12-year-old child with symptomatic vitamin D deficiency rickets with secondary hyperparathyroidism, undergoing general anaesthesia for an urgent orthopaedic procedure. There are numerous risks involved in such a case, such as hypocalcemia, hypophosphatemia, chest and vertebral deformities, restrictive lung disease, difficult intubation and weaning, difficult regional anaesthesia, chronic bone pain, infectious complications and postoperative decreased renal function, all of which require careful preoperative assessment and risk stratification. In elective surgeries, it is important to optimise the metabolic parameters before taking up the case. However, in urgent and emergent procedures like ours, it is imperative to take up the case after informing the parents of the risks involved.

Experimental inoculation of the Botox perineurally of facial nerve and followed by histological assessment for its effect on the zygomatic bone of rabbits

Background: Botox, derived from the toxin produced by Clostridium botulinum, is an attenuated toxin employed as a treatment for cervical dystonia, overactive bladder, strabismus, cerebral palsy, and non-surgical cosmetic procedures. This study aimed to explore the impact of injecting Botox around the facial nerve on the left side of rabbits, specifically focusing on the histological changes in the zygomatic bone. Methods: Twenty-five adult rabbits of a local breed, weighing 1500-1800 gm and of both sexes, underwent a 15-day acclimation period prior to the experiment. The rabbits were randomly assigned to four groups (1st, 2nd, 3rd, and 4th). A singular intramuscular injection of Botox at a dose of 3.5 U/kg was administered to the left facial muscles. The rabbits in each group were then sacrificed at distinct time points after injection: day 10, day 15, day 30, and day 45. All rabbits survived until the end of the experiment, at which point they were euthanized with chloroform. Post-euthanasia, the rabbits' facial zygomatic bones were dissected and subjected to histological assessment. Results: The results indicated notable changes in the zygomatic bone, including prominent vacuoles within the bony matrix, cracks with cellular debris, limited osteocytes, and collagen bundles resembling woven bone. Additionally, in the day 30 group (T3), irregular bone borders and atrophied osteogenic cells were observed, while the day 45 group (T4) exhibited necrotic areas and bone fragments within the bone matrix. Conclusion: In conclusion, Botox, derived from Clostridium botulinum, serves various medical purposes. The study focused on injecting Botox around rabbits' facial nerves to assess its impact on zygomatic bones histologically. Following a controlled injection and specific time intervals, the examined bone samples exhibited distinct changes, providing insights into Botox's effects on bone morphology.

Nanoscale implant anchorage aided by cement line deposition into titanium dioxide nanotubes.

The success of titanium dental implants relies on osseointegration, the load-bearing connection between bone tissue and the device that, in contact osteogenesis, comprises the deposition of bony cement line matrix onto the implant surface. Titanium dioxide nanotubes (NTs) are considered a promising surface for improved osseointegration, yet the mechanisms of cement line integration with such features remains elusive. Herein, we illustrate cement line deposition into NTs on the surface of titanium implants with two underlaying microstructures: a machined surface or a blasted/acid etched surface placed in the tibiae of Wistar rats. After retrieval, scanning electron microscopy of tissue reflected from the implant surface indicated minimal incursion of the cement line matrix into the NTs. To investigate this further, focused ion beam was utilized to prepare cross-sectional samples that could be characterized using scanning transmission electron microscopy. The cement line matrix covered NTs regardless of underlying microstructure, which was further confirmed by elemental analysis. In some instances, cement line infiltration into the NTs was noted, which reveals a mechanism of nanoscale anchorage. This study is the first to demonstrate cement line deposition into titanium NTs, suggesting nano-anchorage as a mechanism for the success of the NT modified surfaces in vivo.

X-ray and MRI Correlation of Bone Tumors Using Histopathology As Gold Standard.

IntroductionBone tumors are a common pathology of the musculoskeletal system being frequently encountered by clinicians. Radiological workup is a mainstay in the diagnostic workup of bone tumors. This study aimed to highlight the importance of plain radiography and MRI in the diagnosis of bone tumors keeping histopathology as a gold standard. It is a descriptive validation study conducted in the Radiology Department of Pakistan Institute of Medical Sciences Islamabad.MethodologyThe study included 92 patients with suspected bone lesions. After taking a complete history and receiving informed written consent. X-rays radiographs and magnetic resonance imaging were performed. X-ray radiograph and magnetic resonance imaging parameters were recorded and compared with the histopathology of lesions as a standard.ResultsThe mean age of patients was 30.50 ± 8.95 years. Of 92 patients examined on X-ray, 51 (55.4%) had lytic lesions, 34 (37.0%) had sclerotic lesions, and seven (7.6 %) had mixed lesions. MRI revealed the location of the lesion. There were 25 (27.2%) bone lesions in diaphysis, 19 (20.7%) in metaphysis, nine (9.8%) at meta-diaphysis, and 32 (34.8 %) in the meta-epiphyseal region. These findings were later on confirmed with histopathological results.ConclusionMRI can differentiate soft-tissue components and periosteal reactions. An X-ray radiograph can provide information about bony matrix and calcifications within tumors. After analysis of imaging findings and histopathological results, it is concluded that these modalities can be used to diagnose bone tumors with high diagnostic accuracy.

An instantly fixable and self-adaptive scaffold for skull regeneration by autologous stem cell recruitment and angiogenesis.

Limited stem cells, poor stretchability and mismatched interface fusion have plagued the reconstruction of cranial defects by cell-free scaffolds. Here, we designed an instantly fixable and self-adaptive scaffold by dopamine-modified hyaluronic acid chelating Ca2+ of the microhydroxyapatite surface and bonding type I collagen to highly simulate the natural bony matrix. It presents a good mechanical match and interface integration by appropriate calcium chelation, and responds to external stress by flexible deformation. Meanwhile, the appropriate matrix microenvironment regulates macrophage M2 polarization and recruits endogenous stem cells. This scaffold promotes the proliferation and osteogenic differentiation of BMSCs in vitro, as well as significant ectopic mineralization and angiogenesis. Transcriptome analysis confirmed the upregulation of relevant genes and signalling pathways was associated with M2 macrophage activation, endogenous stem cell recruitment, angiogenesis and osteogenesis. Together, the scaffold realized 97 and 72% bone cover areas after 12 weeks in cranial defect models of rabbit (Φ = 9 mm) and beagle dog (Φ = 15 mm), respectively.

Supramolecular Peptide Nanofiber Hydrogels for Bone Tissue Engineering: From Multihierarchical Fabrications to Comprehensive Applications.

Bone tissue engineering is becoming an ideal strategy to replace autologous bone grafts for surgical bone repair, but the multihierarchical complexity of natural bone is still difficult to emulate due to the lack of suitable biomaterials. Supramolecular peptide nanofiber hydrogels (SPNHs) are emerging biomaterials because of their inherent biocompatibility, satisfied biodegradability, high purity, facile functionalization, and tunable mechanical properties. This review initially focuses on the multihierarchical fabrications by SPNHs to emulate natural bony extracellular matrix. Structurally, supramolecular peptides based on distinctive building blocks can assemble into nanofiber hydrogels, which can be used as nanomorphology‐mimetic scaffolds for tissue engineering. Biochemically, bioactive motifs and bioactive factors can be covalently tethered or physically absorbed to SPNHs to endow various functions depending on physiological and pharmacological requirements. Mechanically, four strategies are summarized to optimize the biophysical microenvironment of SPNHs for bone regeneration. Furthermore, comprehensive applications about SPNHs for bone tissue engineering are reviewed. The biomaterials can be directly used in the form of injectable hydrogels or composite nanoscaffolds, or they can be used to construct engineered bone grafts by bioprinting or bioreactors. Finally, continuing challenges and outlook are discussed.

Heterotopic Bone Formation in Juvenile Polyp: A Histopathological Entity.

CASE REPORT A 5-year-old boy presented with a 1-month history of blood in stools. The blood was bright red in color, moderate in amount, and associated with intermittent abdominal pain. He had no history of pain during defecation, no constipation, and no alteration in frequency or consistency of stool. Systemic examination revealed only pallor. Local examination of the boy was unremarkable. Colonoscopy examination revealed terminal ileitis with a rectal polyp (Figure 1). A polypectomy was performed, and a specimen was sent for histopathological examination. Grossly, a polypoidal tissue cut section measuring 1.0 × 0.5 × 0.3 cm was firm and greyish white. Microscopic examination showed a polyp with mucosal surface ulceration. Numerous cystically dilated and tortuous crypts filled with inspissated mucin were seen. Intervening lamina propria was edematous and filled with mixed inflammatory cells. One focus showed bony trabeculae rimmed by osteoblast. We diagnosed our patient with juvenile polyp with osseous metaplasia.Figure 1.: Colonoscopy showing polyp in the rectum.Figure 2.: Hematoxylin-eosin stain, ×40 magnification. Juvenile polyp having surface ulceration, and a single black arrow showing osseous metaplasia.Figure 3.: Hematoxylin-eosin stain, ×200 magnification. Juvenile polyp, a single black arrow showing bony matrix, and a single blue arrow showing osteocytes in lacunae.Juvenile polyps are hamartomatous polyps which may undergo osseous metaplasia. In most recent studies, expression of bone (Figures 2 and 3) morphogenic proteins had been observed in the setting of osseous metaplasia.1 Osseous metaplasia, ie, heterotopic bone formation, is an adaptive response to any chronic inflammation. Only a few cases of neoplastic and nonneoplastic gastrointestinal polyps are associated with this extremely rare type of metaplasia.2 It is most commonly seen in the colorectal region in association with epithelial polyps. To the best of our knowledge, only 9 other cases of osseous metaplasia in juvenile polyps have been reported so far worldwide.2 DISCLOSURES Author contributions: M. Mohanty edited the article, reviewed the literature, provided images, and is the article guarantor. N. Pandey wrote the article and reviewed the literature. U. Senapati revised and approved final the article. P. Nath provided images and revised and approved the final article. Acknowledgments: We thank technician Niskara Sahoo for their contribution in good section cutting. Financial disclosure: None to report. Informed consent was obtained for this case report.

Osteogenic-differentiated mesenchymal stem cell-secreted extracellular matrix as a bone morphogenetic protein-2 delivery system for ectopic bone formation

While human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a “bony matrix” containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier.

Hox genes maintain critical roles in the adult skeleton

Hox genes are indispensable for the proper patterning of the skeletal morphology of the axial and appendicular skeleton during embryonic development. Recently, it has been demonstrated that Hox expression continues from embryonic stages through postnatal and adult stages exclusively in a skeletal stem cell population. However, whether Hox genes continue to function after development has not been rigorously investigated. We generated a Hoxd11 conditional allele and induced genetic deletion at adult stages to show that Hox11 genes play critical roles in skeletal homeostasis of the forelimb zeugopod (radius and ulna). Conditional loss of Hox11 function at adult stages leads to replacement of normal lamellar bone with an abnormal woven bone-like matrix of highly disorganized collagen fibers. Examining the lineage from the Hox-expressing mutant cells demonstrates no loss of stem cell population. Differentiation in the osteoblast lineage initiates with Runx2 expression, which is observed similarly in mutants and controls. With loss of Hox11 function, however, osteoblasts fail to mature, with no progression to osteopontin or osteocalcin expression. Osteocyte-like cells become embedded within the abnormal bony matrix, but they completely lack dendrites, as well as the characteristic lacuno-canalicular network, and do not express SOST. Together, our studies show that Hox11 genes continuously function in the adult skeleton in a region-specific manner by regulating differentiation of Hox-expressing skeletal stem cells into the osteolineage.

Deletion of Runx1 in osteoclasts impairs murine fracture healing through progressive woven bone loss and delayed cartilage remodeling.

Conditional deletion of the transcription factor Runt-related transcription factor 1 (Runx1) in myeloid osteoclast precursors promotes osteoclastogenesis and subsequent bone loss. This study posits whether Runx1 regulates clastic cell-mediated bone and cartilage resorption in the fracture callus. We first generated mice, in which Runx1 was conditionally abrogated in osteoclast precursors (LysM-Cre;Runx1F/F ; Runx1 cKO). Runx1 cKO and control mice were then subjected to experimental mid-diaphyseal femoral fractures. Our study found differential resorption of bony and calcified cartilage callus matrix by osteoclasts and chondroclasts within Runx1 cKO calluses, with increased early bony callus resorption and delayed calcified cartilage resorption. There was an increased number of osteoclasts and chondroclasts in the chondro-osseous junction of Runx1 cKO calluses starting at day 11 post-fracture, with minimal woven bone occupying the callus at day 18 post-fracture. LysM-Cre;Runx1F/F mutant mice had increased bone compliance at day 28, but their strength and work to failure were comparable with controls. Taken together, these results indicate that Runx1 is a critical transcription factor in controlling osteoclastogenesis that negatively regulates bone and cartilage resorption in the fracture callus. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1007-1015, 2020.

Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells.

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary “agnostic” DNA microarray and “targeted” NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways involved in osteoblast differentiation, e.g. Wnt/β-catenin signaling, are most active early in the process, while others, e.g. TGFβ/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treatment of conditions characterized by low bone mineral density.

Selection for longer limbs in mice increases bone stiffness and brittleness, but does not alter bending strength.

The ability of a bone to withstand loads depends on its structural and material properties. These tend to differ among species with different modes of locomotion, reflecting their unique loading patterns. The evolution of derived limb morphologies, such as the long limbs associated with jumping, may compromise overall bone strength. We evaluated bone mechanical properties in the Longshanks mouse, which was selectively bred for increased tibia length relative to body mass. We combined analyses of 3D shape and cross-sectional geometry of the tibia, with mechanical testing and bone composition assays, to compare bone strength, elastic properties and mineral composition in Longshanks mice and randomly bred controls. Our data show that, despite being more slender, cortical geometry and predicted bending strength of the Longshanks tibia were similar to controls. In whole bone bending tests, measures of bone bending strength were similar across groups; however, Longshanks tibiae were significantly more rigid, more brittle, and required less than half the energy to fracture. Tissue-level elastic properties were also altered in Longshanks mice, but the bones did not differ from the control in water content, ash content or density. These results indicate that while Longshanks bones are as strong as control tibiae, selection for increased tibia length has altered its elastic properties, possibly through changes in organic bony matrix composition. We conclude that selection for certain limb morphologies, and/or selection for rapid skeletal growth, can lead to tissue-level changes that can increase the risk of skeletal fracture, which in turn may favor the correlated evolution of compensatory mechanisms to mitigate increased fracture risk, such as delayed skeletal maturity.

Osteodystrophia Fibrosa in a Goat - A case report

Osteodystrophia fibrosa is a condition associated with fibrous replacement of the resorbed bony matrix. In India, it is commonly reported in horses, and is most often associated with bran feeding, leading to higher availability of phosphorus and imbalance in the calcium phosphorus ratio. Low calcium or high phosphorus diet can result in secondary nutritional hyperparathyroidism leading to osteodystrophia fibrosa (Ozmen et al., 2017). Bone resorption of calcium occurs associated with low calcium level in circulation. Deformities of the bone manifests associated with the severity of the condition.

Bone resorption and bone metastasis risk

Breast cancer tumors have a tendency to metastasize to the bone. After development of a bone metastasis, the median survival time is 40 months. Currently, little is known about the modifiable risk factors for developing bone metastases in women diagnosed with breast cancer. One possible modifiable risk factor is increased bone resorption. Increased bone resorption is caused by an imbalance between osteoblasts and osteoclasts favoring osteoclast-driven bone resorption. Osteoclast activity results in the release of growth factors from the bony matrix that are requirement for successful breast cancer tumor cell proliferation within the bone. Mice studies have shown that mice that have been genetically engineered to have higher bone mineral density, and thus lower bone resorption, have a decreased incidence of bone metastases. Alternatively, mice genetically engineered to have lower bone mineral density or increased bone resorption have a higher incidence of bone metastases. In human studies, antiosteoporotic drugs have been shown to decrease osteoclast activity and prevent bone metastases. These studies suggest that increased osteoclast activity, which results in low bone mineral density, may be a modifiable risk factor for developing bone metastases in women with breast cancer. Women undergoing chemotherapy for breast cancer develop low bone mineral density in response to the direct effects of chemotherapeutic drugs on bone cells—including osteoclasts, osteoblasts, and osteocytes—and through the decrease in circulating estrogen as a result of chemotherapy-induced ovarian dysfunction. Therefore, it is important for future studies to determine the risk of developing bone metastases associated with increasing bone resorption as measured by low or decreasing bone mineral density in women diagnosed with breast cancer, as well as to determine the best intervention(s) to promote a balance between osteoclasts and osteoblasts to favor osteoblast activity during chemotherapy treatment.

Effect of bone morphogenetic protein on Zn-HAp and Zn-HAp/collagen composite: A systematic in vivo study

Due to good biocompatibility and osteoconductivity, hydroxyapatite (HAp) and its composite with different polymers have been widely investigated for the application in the field of bone tissue engineering. The present study reports the, in vivo performance of zinc doped HAp and HAp/collagen composite (HAC) using bone morphogenetic protein-2. It was done for a span of two months on New Zealand rabbit model. After two months postoperatively, there was no marked inflammatory reaction in experimental groups and control groups. The histological images showed well-formed bony matrix with well differentiated haversian system. From the fluorochrome labeling study, it was observed that higher amount of new bone formed in case of bone morphogenetic protein-2 (BMP-2) loaded Zn-HAp (50%) and HAC (27%) specimens than control. The percentage of new bone formation was significantly higher in case of BMP loaded Zn-HAp group than BMP loaded HAC group. From the SEM images similar trend was observed. As the HAC specimen consists of amorphous phase, it had a negative impact on new bone formation.

Visual perception of the osseous labyrinth rendered from micro-CT scans of the petrous bone.

Grayscale images comparing to the color images may have less of visual information necessary for easy recognition of the anatomical structures. Although micro-CT scanners deliver images of ultra-high resolution, application of false-colors to the rendered structures enhance their visual perception and allow for quick delineation between them and surrounding bony matrix. This paper presents differences of imaging of the osseous structures of the inner ear labyrinth using pseudo-color volume rendering in contrast to grayscale volume rendering of the micro-CT data. Applied procedures of image processing improved significantly delineation between the bony matrix surrounding the cochlea and vestibule rendered in the pseudo-colors than in grayscale.

Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by focusing on differentially supporting formation of cartilaginous and bony matrix by a single cell source in a spatially controlled manner within a single material system. A cell-polymer solution of bovine mesenchymal stem cells and agarose was cast against micromolds of a serpentine network and stacked to produce tissue constructs containing two independent microfluidic networks. Constructs were fluidically connected to two controlled flow loops and supplied with independently tuned differentiation parameters for chondrogenic and osteogenic induction, respectively. Constructs receiving inductive media showed differential gene expression of both chondrogenic and osteogenic markers in opposite directions along the thickness of the construct that was recapitulated at the protein level with respect to collagens I, II, and X. A control group receiving noninductive media showed homogeneous expression of these biomarkers measured in lower concentrations at both the mRNA and protein level. This work represents an important step in the rational design of engineered osteochondral units through establishment of an enabling technology for further optimization of scaffolding formulations and bioprocessing conditions toward the production of commercially viable osteochondral tissue products.

Interface between intramembranous and endochondral ossification in human foetuses.

In the head and neck of human mid-term foetuses, the interface between areas of endochondral ossification and adjacent membranous (intramembranous) ossification is extensive. Using 8 foetal heads at 15-16 weeks, we have demonstrated differences in the matrices and composite cells between these 2 ossification processes, especially in the occipital squama and pterygoid process. Aggrecan-positive cartilage was shown to be invaded by a primitive bony matrix that was negative for aggrecan. At the interface, the periosteum was continuous with the perichondrium without any clear demarcation, but tenascin-c expression was restricted to the periosteum. In contrast, the interface between the epiphysis and shaft of the femur showed no clear localisation of tenascin-c. Versican expression tended to be restricted to the perichondrium. In the pterygoid process, the density of CD34-positive vessels was much higher in endochondral than in membranous ossification. The membranous part of the occipital was considered most likely to contribute to growth of the skull to accommodate the increased volume of the brain, while the membranous part of the pterygoid process seemed to be suitable for extreme flattening under pressure from the pterygoid muscles.