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Background: Surgical reconstruction of congenital heart defects is often limited by the nonresorbable material used to approximate normal anatomy. In contrast, biologic scaffold materials composed of resorbable non-cross-linked extracellular matrix (ECM) have been used for tissue reconstruction of multiple organs and are replaced by host tissue. Preparation of whole organ ECM by decellularization through vascular perfusion can maintain much of the native three-dimensional (3D) structure, strength, and tissue-specific composition. A 3D cardiac ECM (C-ECM) biologic scaffold material would logically have structural and functional advantages over materials such as Dacron™ for myocardial repair, but the in vivo remodeling characteristics of C-ECM have not been investigated to date. Methods and Results: A porcine C-ECM patch or Dacron patch was used to reconstruct a full-thickness right ventricular outflow tract (RVOT) defect in a rat model with end points of structural remodeling function at 16 weeks. The Dacron patch was encapsulated by dense fibrous tissue and showed little cellular infiltration. Echocardiographic analysis showed that the right ventricle of the hearts patched with Dacron were dilated at 16 weeks compared to presurgery baseline values. The C-ECM patch remodeled into dense, cellular connective tissue with scattered small islands of cardiomyocytes. The hearts patched with C-ECM showed no difference in the size or function of the ventricles as compared to baseline values at both 4 and 16 weeks. Conclusions: The C-ECM patch was associated with better functional and histomorphological outcomes compared to the Dacron patch in this rat model of RVOT reconstruction.

Research in cell biology and the development of translational technologies are driven by competition, public expectations, and regulatory oversight, putting these fields at a critical juncture. Success in these fields is quickly becoming dependent on the ability of researchers to identify and isolate specific cell populations from heterogeneous mixtures accurately and efficiently. Many methods for cell purification have been developed, and each has advantages and disadvantages that must be considered in light of the intended application. Current cell separation strategies make use of surface proteins, genetic expression, and physics to isolate specific cells by phenotypic traits. Cell purification is also dependent on the cellular reagents available for use and the intended application, as these factors may preclude certain mechanisms used in the processes of labeling and sorting cells.

The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.

Coordinated activities of multiple mesenchymal cell types contribute to the development of the mammalian skeleton formed through endochondral ossification. Synthesis of a cartilage template by chondrocytes is an obligatory step for the generation of skeletal elements during endochondral ossification. Gene ablation studies have established that Runx2 is an essential transcription factor for bone formation and the differentiation of skeletal cells. However, global gene deletion has failed to discern the tissue- and cell type-specific roles of Runx2. We generated floxed mice to elucidate the Runx2 regulatory control distinctive to cartilage tissue during bone development. Exon 8 of the Runx2 gene was selectively deleted in developing chondrocytes by utilizing Col2a-Cre mice. Cell- and tissue-specific gene recombination was confirmed by β-gal activity in R26R mice. The chondrocyte-specific loss of Runx2 caused failure of endochondral ossification, impaired craniofacial development, dwarfism, and perinatal lethality. Radiographic imaging and histochemical approaches were used to characterize the skeletal phenotype. We conclude that regulatory control of Runx2 in chondrocytes is essential for endochondral ossification, and it is independent of the role of Runx2 in osteoblasts.

Cryogenic transmission electron microscopy (cryo-EM) was used to explore the self-assembly of recombinant murine amelogenin (rM179) in vitro.Our cryo-EM data showed that amelogenin self-assembly is a strongly pH-dependent process. At pH 4.4 the main fraction of the protein exists in a monomeric form, although some peculiar structures consisting of chains of monomers were also observed. At pH 5.8 large nanospheres comprising ring-like structures ∼50 nm in diameter were the most abundant particle class. Similarly, at pH 8.0 amelogenins self-assembled into ring-like oligomers of different sizes, which subsequently assembled into nanospheres 15–20 nm in diameter. Furthermore, at pH 7.2, which is close to a physiological pH, branched chains of nanospheres were observed. Our results show that amelogenin assembly is a multistep hierarchical process and provides new insight into the control of enamel mineralization.

Fibromodulin (Fmod) is a keratan sulfate small leucine-rich proteoglycan which is enriched in bones and teeth. In order to determine its functions on bone and tooth mineralization we characterized the phenotype of Fmod-deficient (Fmod-KO) mice using a new-generation microfocus computerized tomography system (micro-CT) and software allowing advanced visualization of 3-D data. Three-week-old and 10- week-old Fmod-KO mandibles and teeth were compared with those of age-matched wild-type (WT) mice. In both young and mature mice the Fmod-KO mandibles were hypomineralized, especially the posterior (proximal) part of the mandible as it appeared to be the main target of the molecule deficiency whereas less extensive alterations were found in the alveolar bone. In transverse sections, larger marrow spaces were observed in the Fmod-KO mice compared with age-matched young or mature WT mice. Quantitative evaluation of the pulp volume of the first molar and 3-D reconstructions suggested that dentinogenesis was diminished in 3-week-old Fmod-KO teeth. In contrast, increased dentin formation was found in 10-week-old Fmod-KO mice and it was accompanied by a reduced pulp volume. Thus, the differential effects of Fmod deficiency on bones and teeth appear to diverge in adult mice. This may result from the previously reported differences in the molecular weight of Fmod in the 2 tissues or from compensatory mechanisms due to the overexpression of DSP and DMP-1 in the dental pulp of Fmod-KO. It is also possible that a single molecule plays diverging roles in a tissue-specific or region-specific manner.

N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (–P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(–P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

Excessive systemic exposure to fluoride (F) can lead to disturbances in bone homeostasis and dental enamel development. We have previously shown strain-specific responses to F in the development of dental fluorosis (DF) and in bone formation/mineralization. The current study was undertaken to further investigate F responsive variations in bone metabolism and to determine possible relationships with DF susceptibility. Seven-week-old male mice from FVB/NJ, C57BL/6J, C3H/HeJ, A/J, 129S1/SvImJ, AKR/J, DBA/2J, and BALB/cByJ inbred strains were exposed to NaF (0 or 50 ppm as F–) in drinking water for 60 days. Sera were collected for F, Ca, Mg, PO4, iPTH, sRANKL, and ALP levels. Bone marrow cells were subjected to ex vivo cell culture for osteoclast potential and CFU colony assays (CFU-fibroblast, CFU-osteoblast, CFU-erythrocyte/granulocyte/macrophage/megakaryocyte, CFU-granulocyte/macrophage, CFU-macrophage, and CFU-granulocyte). Femurs and vertebrae were subjected to micro-CT analyses, biomechanical testing, and F, Mg, and Ca content assays. DF was evaluated using quantitative fluorescence and clinical criteria. Strain-specific responses to F were observed for DF, serum studies, ex vivo cell culture studies, and bone quality. Among the strains, there were no patterns or significant correlations between DF severity and the actions of F on bone homeostasis (serum studies, ex vivo assays, or bone quality parameters). The genetic background continues to play a role in the actions of F on tooth enamel development and bone homeostasis. F exposure led to variable phenotypic responses between strains involving dental enamel development and bone metabolism.

A single recurrent mutation in the regulatory subdomain of a bone morphogenetic protein type I receptor kinase has been linked to heterotopic ossification in classic fibrodysplasia ossificans progressiva (FOP). As a result of a substitution at 1 residue by only 1 other side chain (Arg206His) in just 1 of the 4 type I BMP receptors (ALK2/ACVR1), soft connective tissues progressively metamorphose through an endochondral process into cartilage that is replaced by bone. The substitution of arginine for histidine, also a basic residue yet with the singular property of ionization/protonation over the physiological pH range, led to the hypothesis of an aberrant, pH-sensitive switch mechanism for the ligand-independent activation of BMP signaling through the mutant receptor kinase in patients presenting with classic FOP. To test a potential aspect of the putative pH-dependent mechanism, i.e. loss of autoinhibition of the kinase mediated by the inhibitory protein FKBP12, in vitrointeraction analyses with purified wild-type and R206H ALK2 kinase and FKBP12 proteins were performed. Interactions between the kinases and inhibitory proteins were analyzed qualitatively and quantitatively by native gel electrophoresis and HPLC size exclusion chromatography and with an optical biosensor (Octet; ForteBio). Binding of inhibitory protein by the R206H mutant was diminished 3-fold relative to the wild type kinase at a physiological pH, yet below this value (

Dental enamel development occurs in stages as observed by the changing morphology of the ameloblasts that are responsible for enamel formation. During the secretory stage of development, proteins including MMP20 are secreted into the enamel matrix. MMP20 is required for proper enamel formation as mutation of the Mmp20 gene causes autosomal recessive amelogenesis imperfecta. Here, we examined in detail the morphology of the Mmp20-null ameloblast cell layer. Intriguingly, we found that the Mmp20-null mouse secretory stage ameloblasts retract their Tomes’ processes as if preparing to enter the maturation stage but later reextend their Tomes’ processes as if resuming the secretory stage. We also demonstrated that MMP20 cleaves epithelial cadherin, i.e. E-cadherin. Cadherins are transmembrane proteins with extracellular domains that provide adhesive contacts between neighboring cells. Their intracellular domains bind to the cell cytoskeleton through catenins, including β-catenin. When specific MMPs cleave the cadherin extracellular domain, β-catenin is released and may locate to the cell nucleus as a transcription factor. Therefore, MMP20 may influence ameloblast developmental progression through hydrolysis of cadherin extracellular domains with associated release of transcription factor(s).

Enamel matrix proteins, including the most abundant amelogenin and lesser amounts of enamelin, ameloblastin, and proteinases, play vital roles in controlling crystal nucleation and growth during enamel formation. The cooperative action between amelogenin and the 32-kDa enamelin is critical to regulating the growth morphology of octacalcium phosphate crystals. Using biophysical methods, we investigated the interaction between the 32-kDa enamelin and recombinant pig amelogenin 148 (rP148) at pH 6.5 in phosphate-buffered saline (PBS). Dynamic light scattering results showed a trend of increasing particle size in the mixture with the addition of enamelin to amelogenin. Upon addition of the 32-kDa enamelin, the shift and intensity decrease in the ellipticity minima of rP148 in the circular dichroism spectra of rP148 illustrated a direct interaction between the 2 proteins. In the fluorescence spectra, the maximum emission of rP148 was blue shifted from 335 to 333 nm in the presence of enamelin as a result of complexation of the 2 proteins. Our results demonstrate that the 32-kDa enamelin has a close association with amelogenin at pH 6.5 in PBS buffer. Our present study provides novel insights into the possible cooperation between enamelin and amelogenin in macromolecular coassembly and in controlling enamel mineral formation

In this study, patterns of activity in the soleus (Sol) and tibialis anterior (TA) muscles and hindlimb kinematics were evaluated during slope walking in rats after transection and surgical repair either of the entire sciatic nerve (Sci group) or of its two branches separately, the tibial and common fibular nerves (T/CF group). With the latter method, axons from the tibial and common fibular nerves could not reinnervate targets of the other nerve branch after injury, reducing the opportunity for misdirection. Activity in the TA shifted from the swing phase in intact rats to nearly the entire step cycle in both injured groups. Since these changes occur without misdirection of regenerating axons, they are interpreted as centrally generated. Sol activity was changed from reciprocal to that of TA in intact rats to coactivate with TA, but only in the Sci group rats. In the T/CF group rats, Sol activity was not altered from that observed in intact rats. Despite effects of injury that limited foot movements, hindlimb kinematics were conserved during downslope walking in both injury groups and during level walking in the T/CF group. During level walking in the Sci group and during upslope walking in both groups of injured rats, the ability to compensate for the effects of the nerve injury was less effective and resulted in longer limb lengths held at more acute angles throughout the step cycle. Changes in limb movements occur irrespective of axon misdirection and reflect compensatory changes in the outputs of the neural circuits that drive locomotion.

Current models and concepts of motor control represent the limb as a neuro-musculoskeletal system and rarely include other potentially important supporting tissues such as fascia and adipose tissue. It is possible that a normal complement of adipose tissue could contribute to the viscoelastic properties of supporting limbs and enhance stability during locomotion. The purpose of this study was to determine if the popliteal fat pad plays a role in locomotion in the cat. It is hypothesized that the fat pad limits flexion and reduces angular acceleration of the included hip, knee and ankle joints in the sagittal plane throughout the step cycle. 3D kinematics from 3 spontaneously locomoting decerebrate cats both before and after lipectomy were recorded during treadmill walking. Four time points throughout the step cycle were chosen for angular acceleration analysis: mid-stance, paw off, mid-swing and peak deceleration at the end of the re-extension of the knee. Significant increases in maximum angular acceleration for the hip, knee and ankle joints at these time points were observed. No significant increase in range of motion was found across all 3 included angles after lipectomy. Therefore, the hypothesis that the popliteal fat pad acts to decrease the angular acceleration is supported by these findings. The data indicate that the popliteal fat pad contributes to the damping component of the viscoelastic properties of the limb. These results may be applied to models of the hindlimb and knowledge of the effects of obesity on movement.

Transforming growth factor-β (TGF-β) normally inhibits breast cancer development by preventing mammary epithelial cell (MEC) proliferation, by inducing MEC apoptosis, and by creating cell microenvironments that maintain MEC homeostasis and prevent their uncontrolled growth and motility. Mammary tumorigenesis elicits dramatic alterations in MEC architecture and microenvironment integrity, which collectively counteract the tumor-suppressing activities of TGF-β and enable its stimulation of breast cancer invasion and metastasis. How malignant MECs overcome the cytostatic actions imposed by normal microenvironments and TGF-β, and how abnormal microenvironments conspire with TGF-β to stimulate the development and progression of mammary tumors remains largely undefined. These knowledge gaps have prevented science and medicine from implementing treatments effective in simultaneously targeting abnormal cellular microenvironments, and in antagonizing the oncogenic activities of TGF-β in developing and progressing breast cancers. c-Abl is a ubiquitously expressed nonreceptor protein tyrosine kinase that essentially oversees all aspects of cell physiology, including the regulation of cell proliferation, migration and adhesion, as well as that of cell survival. Thus, the biological functions of c-Abl are highly reminiscent of those attributed to TGF-β, including the ability to function as either a suppressor or promoter of tumorigenesis. Interestingly, while dysregulated Abl activity clearly promotes tumorigenesis in hematopoietic cells, an analogous role for c-Abl in regulating solid tumor development, including those of the breast, remains controversial. Here, we review the functions of c-Abl in regulating breast cancer development and progression, and in alleviating the oncogenic activities of TGF-β and its stimulation of epithelial-mesenchymal transition during mammary tumorigenesis.

Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 μm average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 ± 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.

The overall goal of the investigation was to examine autophagy in the growth plate and to ascertain how this process was regulated. Herein, we show that in the postmitotic maturing zone of the growth plate, chondrocytes express an autophagic phenotype. This robust and particulate immunohistochemical response provides direct evidence that autophagy is a new and transient stage in the chondrocyte maturation pathway. We found that induction of autophagy was regulated by mTOR, a sensor of cellular metabolism. When mTOR was inhibited, changes in LC3 fluorescence indicated that this kinase regulated development of the autophagic state. To determine if AMP kinase was required for chondrocyte autophagy, we suppressed its expression in N1511 cells using siRNA technology. When these cells were serum starved, a condition that triggers autophagy, there was no change in LC3 distribution. This result confirmed that AMP kinase was required for the induction of the autophagic response. Based on the 2 studies described above, and our previous observation that HIF-1 is required for the induction of autophagy, we put forward the hypothesis that autophagy is regulated by the activities of AMP kinase and mTOR in a HIF-1-dependent manner. Once autophagy is activated, the postmitotic chondrocytes would be expected to remain viable in their unique microenvironment and complete their life cycle.

Familial hypophosphatemic rickets is transmitted in most cases as an X-linked dominant trait and results from the mutation of the PHEX gene predominantly expressed in osteoblast and odontoblast. Patients with rickets have been reported to display important dentin defects. Our purpose was to explore the structure, composition and distribution of noncollagenous proteins (NCPs) of hypophosphatemic dentin. We collected teeth from 10 hypophosphatemic patients whose mineralization occurred either in a hypophosphatemic environment or in a corrected phosphate and vitamin environment. Teeth were examined by scanning electron microscopy, immunohistochemistry and Western blot analysis. An abnormal distribution (accumulation in interglobular spaces) and cleavage of the NCPs and particularly of matrix extracellular phosphoglycoprotein were observed in deciduous dentin. In contrast, it was close to normal in permanent dentin mineralized under corrected conditions. In conclusion, dentin mineralization in a corrected phosphate and vitamin D environment compensates the adverse effect of PHEX mutation.