The Liberfarb syndrome, a multisystem disorder affecting eye, ear, bone, and brain development, is caused by a founder pathogenic variant in thePISD gene.

PURPOSE: We observed four individuals in two unrelated but consanguineous families from Portugal and Brazil affected by early-onset retinal degeneration, sensorineural hearing loss, microcephaly, intellectual disability, and skeletal dysplasia with scoliosis and short stature. The phenotype precisely matched that of an individual of Azorean descent published in 1986 by Liberfarb and coworkers. METHODS: Patients underwent specialized clinical examinations (including ophthalmological, audiological, orthopedic, radiological, and developmental assessment). Exome and targeted sequencing was performed on selected individuals. Minigene constructs were assessed by quantitative polymerase chain reaction (qPCR) and Sanger sequencing. RESULTS: Affected individuals shared a 3.36-Mb region of autozygosity on chromosome 22q12.2, including a 10-bp deletion (NM_014338.3:c.904-12_904-3delCTATCACCAC), immediately upstream of the last exon of the PISD (phosphatidylserine decarboxylase) gene. Sequencing of PISD from paraffin-embedded tissue from the 1986 case revealed the identical homozygous variant. In HEK293T cells, this variant led to aberrant splicing of PISD transcripts. CONCLUSION: We have identified the genetic etiology of the Liberfarb syndrome, affecting brain, eye, ear, bone, and connective tissue. Our work documents the migration of a rare Portuguese founder variant to two continents and highlights the link between phospholipid metabolism and bone formation, sensory defects, and cerebral development, while raising the possibility of therapeutic phospholipid replacement.

Disordered vertebral and rib morphology in pudgy mice. Structural relationships to human scoliosis.

Normal and abnormal vertebral development have been studied over the past 200 years at increasing levels of resolution as techniques for biological investigation have improved. Disordered development of the axial skeleton from the early embryonic period on leads to structurally malformed vertebrae and intervertebral discs and ribs causing the severe deformities of scoliosis, kyphosis, and kyphoscoliosis. Developmental malformation of the axial skeleton therefore has led to considerable biological and clinical interest. This work will detail our studies on the structural deformities of the vertebral column and adjacent ribs in the pudgy mouse [1] caused by mutations in the delta-like 3 (Dll3) gene of the Notch family [2]. While gene abnormalities in the pudgy mouse have been outlined, there has been no in-depth assessment of the histopathology of the pudgy vertebral and rib abnormalities that this study will provide. In addition, although congenital scoliosis has been recognized as a clinical problem since the mid-nineteenth century (1800s) [3] and accurately defined by radiography since the early twentieth century (1900s) [4-6], there have been few detailed histopathologic studies of human cases. We will also relate our histopathologic findings in the pudgy mouse to the histopathology of human vertebral and rib malformations in clinical cases of congenital scoliosis, one of which we defined in detail previously [7].

Materials and Methods.

Mice obtained for this study were products of pudgy breeding pairs from Jackson Laboratories, Bar Harbor, Maine. For the affected pudgy mice, heterozygous unaffected littermates served as controls. An affected pudgy mouse (pu/pu) can be identified at birth since it is approximately three-quarters the length of its non-affected littermates (pu/+) and has a markedly shortened, twisted tail. The mice were sacrificed by intraperitoneal injections of sodium pentobarbital. Vertebral and rib assessments were performed in 68 mice, 37 affected (pu/pu) and 31 non-affected (pu/+) age-matched siblings from the late embryo to 3 months of age. There were eight sets of births (litters) in which two or more of the sibling littermates were affected, allowing for a comparison of rib and vertebral anomalies in pudgy mice from the same mother and same pregnancy as well as with all other pudgy mice.

Results.

Table 3.1 outlines ages of pudgy and non-affected mice, sibling groups with two or more littermates affected, and types of structural studies performed in each mouse. Table 3.2 outlines the age distribution of the studies.

Conclusions.

The abnormal findings in the pudgy mouse are remarkably similar to those in the severe case of human congenital scoliosis such as seen in the spondylocostal dysplasias. The pudgy mouse is an excellent model to study: (i) how gene mutations translate into three-dimensional structural abnormalities and (ii) which other factors contribute to the final deformities.

Discussion.

Each of the radiographic, histologic, whole mount, and three-dimensional computerized reconstruction techniques throws specific light on the vertebral, intervertebral disc, and rib abnormalities in the development of the pudgy mouse. The end result demonstrates a failure of both normal formation and normal segmentation in the pudgy mouse recognized now as a recessive genetic disorder in which mutations in the Delta-like 3 gene (Dll3) have been defined [2]. The variable appearance of the vertebral and rib abnormalities from mouse to mouse, including in particular the variable appearance in affected littermates, indicates that the gene abnormality alone does not account solely for the deformities seen. Once the gene abnormality is expressed early in embryogenesis, secondary effects, presumably by mechanisms of epigenetics, appear to play a significant role in outlining the abnormal pattern. The pudgy mouse and other genetically triggered axial developmental abnormalities represent excellent models to help unravel pathogenetic mechanisms whereby gene abnormalities are translated into three-dimensional structural abnormalities.

Anesthetic management of 877 pediatric patients undergoing muscle biopsy for neuromuscular disorders: a 20-year review.

BACKGROUND: The objectives are to review the anesthetic management and anesthetic-related adverse events in patients undergoing muscle biopsy for a broad spectrum of neuromuscular disorders (NMD). AIM: The study aims to assess the hypothesis that perceived awareness of potential anesthesia-induced hyperkalemia and MH in patients with NMD reduces the frequency of such events. METHODS: A 20-year retrospective review of 877 consecutive patients undergoing muscle biopsy to establish diagnoses of NMD has been performed. Patients were categorized prebiopsy into six groups: M (myopathy and muscular dystrophy), MM (mitochondrial or metabolic myopathy), N (neurodegenerative, peripheral neuropathy or spinal muscular atrophy disorder), D (dermatomyositis), C (cardiomyopathy), or S (seizure disorder). Data were collected for demographics, anesthetic management, pre- and postoperative anesthesia-induced muscle injury, postbiopsy histopathologic diagnosis, and concordance comparisons between pre- and postbiopsy diagnoses. RESULTS: There were 513 males (58.5%) and 364 females (41.5%) (1.4:1) with 137 individuals (15.6%) operated on under 1 year of age and two-thirds by 6 years of age. NMD diagnosis was reached in 409 (46.6%) while 468 (53.4%) had no specific pathology. No patients exhibited signs of anesthesia-induced muscle injury (malignant hyperthermia, rhabdomyolysis, cardiac arrest, or postoperative deterioration of weakness). MM was the largest group pre biopsy (367, 41.8%). Anesthetic agents were: nitrous oxide in 657 (74.9%); volatile agents in 139 (15.8%); intravenous agents in 836 (95.3%) (primarily propofol, midazolam, and fentanyl); nondepolarizing muscle relaxants in 404 (46.1%); and regional anesthesia in 112 (12.8%) [most commonly spinal anesthesia in 80 (71.4%)]. Comparing preoperative diagnostic category with postoperative diagnosis, there was a concordance of 78% (319/409) between the two for cases with a definitive diagnosis and 89.7% (787/877) for all cases. CONCLUSIONS: In this retrospective study, no patient exhibited signs or symptoms of hyperkalemia or MH probably because the incidence is very low and becomes even less likely due to the selection of the various anesthetic agents and strategies administered.

The cn/cn dwarf mouse. Histomorphometric, ultrastructural, and radiographic study in mutants corresponding to human acromesomelic dysplasia Maroteaux type (AMDM).

BACKGROUND: The cn/cn dwarf mouse is caused by a loss-of-function mutation in the natriuretic peptide receptor 2 (NPR-2) gene which helps positively regulate endochondral longitudinal bone growth. The gene mutation corresponds to that in the human skeletal dysplasia Acromesomelic Dysplasia Maroteaux type (AMDM). This study assesses histomorphometric, ultrastructural and radiographic correlates of the growth abnormality. METHODS: Ten litters of cn/cn and cn/+littermates at ages ranging from 2.5 to 6.5 weeks were studied by skeletal radiographs, histomorphometry and physeal ultrastructure. Skeletal radiographs were done on 2 cn/cn and 2 cn/+littermates at 5 weeks of age. Humeral, femoral, and tibial lengths were measured from 34 intact bones (17 cn/cn, 17 cn/+) at 2.5 to 6.5 weeks. Growth plate histomorphometry in 50 bones (26 cn/cn and 24 cn/+) determined the hypertrophic zone/entire physeal cartilage ratios in 204 sections (87 cn/+, 117 cn/cn) at 3 time periods (2.5-3, 4-4.5, and 6-6.5 weeks). Electron microscopy assessed 6 cn/cn and 6 cn/+age and site-matched physeal cartilage. RESULTS: Cn/cn mice were two thirds the size of the cn/+. Cn/cn bones were normal in shape or only minimally deformed except for the radius with mid-diaphyseal bowing. Length ratios of cn/cn humeri, femurs, and tibias were a mean of 0.65 (± 0.03, n = 34, 17 ratios) compared to cn/+bones. The main physeal abnormality was a markedly shortened hypertrophic zone with the ratio of hypertrophic zone to entire physis 0.17 (± 0.063) in the cn/cn and 0.30 (± 0.052) in the cn/+mice. Ratio assessments were similar comparing humeral, femoral, and tibial growth plates as were ratios from each of the 3 time periods. Ultrastructural assessments from the resting zone to the lower hypertrophic zone-metaphyseal junction showed no specific individual cell abnormalities in cn/cn compared to cn/+physes. CONCLUSIONS: The disorder causes a shortened physeal hypertrophic zone but normal ultrastructure of cn/cn chondrocytes points to abnormality primarily affecting the hypertrophic zone rather than a structural cell or matrix synthesis problem.

Pudgy mouse rib deformities emanate from abnormal paravertebral longitudinal cartilage/bone accumulations.

The pudgy (pu/pu) mouse, caused by a recessive mutation in the Notch family Delta like-3 gene (Dll3), has severe rib, vertebral body and intervertebral disc abnormalities. Using whole-mount preparations and serial histologic sections we demonstrate: 1) localized paravertebral longitudinal cartilage/bone accumulations (PVLC/BAs) invariably associated with branched, fused and asymmetrically spaced ribs that emanate from it laterally; 2) abnormal rib formation immediately adjacent to abnormal vertebral body and intervertebral disc formation in asymmetric right/left fashion; and 3) patterns of rib deformation that differ in each mouse. Normal BALB/c embryo and age-matched non-affected pu/+ mice assessments allow for pu/pu comparisons. The Dll3 Notch family gene is involved in normal somitogenesis via the segmentation clock mechanism. Although pathogenesis of rib deformation is initially triggered by the Dll3 gene mutation, these findings of abnormal asymmetric costo-vertebral region structure imply that differing patterns cannot be attributed to this single gene mutation alone. All findings implicate a dual mechanism of malformation: the Dll3 gene mutation leading to subtle timing differences in traveling oscillation waves of the segmentation clock and further subsequent misdirection of tissue formation by altered chemical reaction-diffusion and epigenetic landscape responses. PVLC/BAs appear as primary supramolecular structures underlying severe rib malformation associated both with time-sensitive segmentation clock mutations and subsequent reactions.

Lamellar thickness measurements in control and osteogenesis imperfecta human bone, with development of a method of automated thickness averaging to simplify quantitation.

Lamellar bone that forms in moderate and severe osteogenesis imperfecta (OI) is composed of structurally irregular lamellae compared to those in control bone. OI and control cortical bone fragments were prepared for light microscopy in standardized fashion: decalcified, embedded in plastic, sectioned and stained with toluidine blue. Polarization light microscopy (PLM) was used to demonstrate and quantify bright and dark lamellar thicknesses in cortical bone fragments from 5 patients with moderate to severe OI in whom type I collagen structural/molecular defects were detected and in control bone from 5 patients. Rigid selection criteria identified lamellar regions for quantification. Thicknesses of bright and dark lamellae were measured manually at 20X magnification using a histomorphometric image analysis system. A method of automated thickness averaging was developed to determine lamellar thicknesses from PLM images to make measurement faster. Our study demonstrates, for the first time, that in OI bone from patients with type I collagen structural/molecular defects mean lamellar thickness measurements (along with the bright and dark lamellar thicknesses) were less than those in control bone by statistically highly significant differences. The mean value for bright lamellae was less than that for dark lamellae in both control and OI bone. The ratio of mean values for bright/dark lamellar thicknesses was the same in control and OI bone. The automated method obtained similar results to the manual method. Lamellar bone in moderate and severe OI with type I collagen defects is composed of thinner and less structurally regular lamellae than those in control bone. This finding indicates that lamellar thickness measurements can be helpful in assessing the effect of specific collagen and collagen-related mutations on OI bone synthesis and warrant inclusion in research and clinical histomorphometric assessments.

Automated measurement of lamellar thickness in human bone using polarized light microscopy.

Lamellar bone formed in individuals with moderate and severe osteogenesis imperfecta (OI) is often composed of lamellae that are structurally abnormal. Measuring the thickness of these lamellae can be helpful in assessing the effect of specific collagen and collagen-related mutations on OI bone synthesis. Manual measurement of lamellar thicknesses in large quantities is very time consuming. The method for automated measurement described in this article utilizes an image processing script to identify the average thickness of multiple lamellae automatically from histologic images of bone. This allows for faster measurements that are less prone to human error and can account for variability in the thickness of a lamella along its length.•OI and control bone samples are prepared per the glycol methacrylate resin (JB-4 plastic) technique and viewed using polarized light microscopy.•Ideal bone regions for measurement are identified using specific qualitative criteria designed to ensure uniform and accurate thickness measurements.•The method was validated with dataset containing 211 lamellae from control bone and 212 lamellae from OI bone.

Rapid reversal of uremic neuropathy following renal transplantation in an adolescent.

Clinical and pathologic studies on adults with uremic neuropathy are numerous, but less is known about this disorder in children and adolescents. We report the clinical, electrophysiologic, and pathologic findings in an adolescent female with uremic neuropathy. Electrophysiologic findings were consistent with a primarily axonal sensorimotor polyneuropathy. Sural nerve biopsy revealed areas of focal depletion in myelin sheaths and loss of axons. Axonal degeneration with secondary myelin changes appears to be the characteristic pathology in this case, one of the youngest to our knowledge for which nerve biopsy data are available. Our patient experienced dramatic recovery after renal transplantation, similar to the reports of older patients.

Computational model of endochondral ossification: Simulating growth of a long bone.

Mechanical loading is a crucial factor in joint and bone development. Using a computational model, we investigated the role of mechanics on cartilage growth rate, ossification of the secondary center, formation of the growth plate, and overall bone shape. A computational algorithm was developed and implemented into finite element models to simulate the endochondral ossification for symmetric and asymmetric motion in a generic diarthrodial joint. Under asymmetric loading condition the secondary center ossifies asymmetrically leaning toward the external load and results in tilted growth plate. Also the mechanics seems to have greater influence in the early onset of the ossification of the secondary center rather than later progression of the center. While previous models have simulated select stages of skeletal development, our model can simulate growth and ossification during the entirety of post-natal development. Such computational models of skeletal development may provide insight into specific loading conditions that cause bone and joint deformities, and the required timing for rehabilitative repair.

Histopathology of osteogenesis imperfecta bone. Supramolecular assessment of cells and matrices in the context of woven and lamellar bone formation using light, polarization and ultrastructural microscopy.

Diaphyseal long bone cortical tissue from 30 patients with lethal perinatal Sillence II and progressively deforming Sillence III osteogenesis imperfecta (OI) has been studied at multiple levels of structural resolution. Interpretation in the context of woven to lamellar bone formation by mesenchymal osteoblasts (MOBLs) and surface osteoblasts (SOBLs) respectively demonstrates lamellar on woven bone synthesis as an obligate self-assembly mechanism and bone synthesis following the normal developmental pattern but showing variable delay in maturation caused by structurally abnormal or insufficient amounts of collagen matrix. The more severe the variant of OI is, the greater the persistence of woven bone and the more immature the structural pattern; the pattern shifts to a structurally stronger lamellar arrangement once a threshold accumulation for an adequate scaffold of woven bone has been reached. Woven bone alone characterizes lethal perinatal variants; variable amounts of woven and lamellar bone occur in progressively deforming variants; and lamellar bone increasingly forms rudimentary and then partially compacted osteons not reaching full compaction. At differing levels of microscopic resolution: lamellar bone is characterized by short, obliquely oriented lamellae with a mosaic appearance in progressively deforming forms; polarization defines tissue conformations and localizes initiation of lamellar formation; ultrastructure of bone forming cells shows markedly dilated rough endoplasmic reticulum (RER) and prominent Golgi bodies with disorganized cisternae and swollen dispersed tubules and vesicles, structural indications of storage disorder/stress responses and mitochondrial swelling in cells with massively dilated RER indicating apoptosis; ultrastructural matrix assessments in woven bone show randomly oriented individual fibrils but also short pericellular bundles of parallel oriented fibrils positioned obliquely and oriented randomly to one another and in lamellar bone show unidirectional fibrils that deviate at slight angles to adjacent bundles and obliquely oriented fibril groups consistent with twisted plywood fibril organization. Histomorphometric indices, designed specifically to document woven and lamellar conformations in normal and OI bone, establish ratios for: i) cell area/total area X 100 indicating the percentage of an area occupied by cells (cellularity index) and ii) total area/number of cells (pericellular matrix domains). Woven bone is more cellular than lamellar bone and OI bone is more cellular than normal bone, but these findings occur in a highly specific fashion with values (high to low) encompassing OI woven, normal woven, OI lamellar and normal lamellar conformations. Conversely, for the total area/number of cells ratio, pericellular matrix accumulations in OI woven are smallest and normal lamellar largest. Since genotype-phenotype correlation is not definitive, interposing histologic/structural analysis allowing for a genotype-histopathologic-phenotype correlation will greatly enhance understanding and clinical management of OI.

Inefficient dystrophin expression after cord blood transplantation in Duchenne muscular dystrophy.

We report a boy who received two allogeneic stem cell transplantations from umbilical cord donors to treat chronic granulomatous disease (CGD). The CGD was cured after the second transplantation, but 2.5 years later he was diagnosed with Duchenne muscular dystrophy (DMD). Examinations of his DNA, muscle tissue, and myoblast cultures derived from muscle tissue were performed to determine whether any donor dystrophin was being expressed. The boy was found to have a large-scale deletion on the X chromosome that spanned the loci for CYBB and DMD. The absence of dystrophin led to muscle histology characteristic of DMD. Analysis of myofibers demonstrated no definite donor cell engraftment. This case suggests that umbilical cord-derived hematopoietic stem cell transplantation will not be efficacious in the therapy of DMD without additional interventions that induce engraftment of donor cells in skeletal muscle.

Accuracy of preoperative electrocardiographic and chest radiographic screening for prediction of left ventricular dysfunction in patients with suspected neuromuscular disorders.

BACKGROUND: We sought to determine the reliability of electrocardiography (ECG) and chest radiography (CXR) in predicting left ventricular (LV) dysfunction in patients with suspected neuromuscular disorders (NMDs) undergoing preanesthetic evaluation for muscle biopsy. METHODS: In this retrospective study, 255 patients with a preliminary diagnosis of NMDs based on history, physical examination, and laboratory testing underwent preanesthetic screening before muscle biopsy. The screening included various combinations of ECG, CXR, and transthoracic echocardiography (Echo) to assess perioperative risk associated with potentially undiagnosed LV dysfunction. Multivariate logistic regression analysis was applied to ascertain whether CXR and ECG were independently predictive of LV dysfunction. In addition, receiver-operating characteristic curve analysis was used to assess the diagnostic accuracy of each test and the combination of CXR and ECG in differentiating LV dysfunction from normal function based on Echo "gold standard" data. RESULTS: The study consisted of 255 patients who had a transthoracic Echo, and among these patients, 235 had CXR and 237 had ECG. Forty-four patients were diagnosed by transthoracic Echo to have LV dysfunction (17.3%). Of the 255 patients in the study population, 24 were found to have mild LV dysfunction (9.4%) and 20 had moderate to severe LV dysfunction (7.8%) on Echo. With Echo providing the definitive standard for the diagnosis of LV dysfunction, we found that a CXR alone was predictive in 37% of cases of LV dysfunction, an ECG alone was predictive in 14% of cases, and the combination of both was predictive in 81% of cases. The combination of ECG and CXR test offered the highest diagnostic accuracy (area under the curve of 0.95, P < 0.0001) for differentiating moderate to severe LV dysfunction from normal LV function. CONCLUSIONS: In patients with suspected neuromuscular disease, CXR and ECG provided low independent diagnostic prediction for the presence or absence of LV dysfunction. The combination of both tests can identify cardiomyopathy with relatively high accuracy in children with suspected NMDs independent of age and gender, particularly in patients with moderate to severe LV dysfunction. Although our findings suggest that combination ECG and CXR screening is a reliable means of detecting LV dysfunction, this approach fails to differentiate the severity or type of cardiomyopathy that may exist. Therefore, the decision to obtain a perioperative Echo before muscle biopsy should involve careful consideration of the disease suspected, ECG and CXR results, laboratory studies, patient age, physical examination, and family history.

Direct Reprogramming of Mouse Fibroblasts into Functional Osteoblasts.

Although induced pluripotent stem cells hold promise as a potential source of osteoblasts for skeletal regeneration, the induction of pluripotency followed by directed differentiation into osteoblasts is time consuming and low yield. In contrast, direct lineage reprogramming without an intervening stem/progenitor cell stage would be a more efficient approach to generate osteoblasts. We screened combinations of osteogenic transcription factors and identified four factors, Runx2, Osx, Dlx5, and ATF4, that rapidly and efficiently reprogram mouse fibroblasts derived from 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP) transgenic mice into induced osteoblast cells (iOBs). iOBs exhibit osteoblast morphology, form mineralized nodules, and express Col2.3GFP and gene markers of osteoblast differentiation. The global transcriptome profiles validated that iOBs resemble primary osteoblasts. Genomewide DNA methylation analysis demonstrates that within differentially methylated loci, the methylation status of iOBs more closely resembles primary osteoblasts than mouse fibroblasts. We further demonstrate that Col2.3GFP+ iOBs have transcriptome profiles similar to GFP+ cells harvested from Col2.3GFP mouse bone chips. Functionally, Col2.3GFP+ iOBs form mineralized bone structures after subcutaneous implantation in immunodeficient mice and contribute to bone healing in a tibia bone fracture model. These findings provide an approach to derive and study osteoblasts for skeletal regeneration. © 2019 American Society for Bone and Mineral Research.

Molecular differentiation in epiphyseal and physeal cartilage. Prominent role for gremlin in maintaining hypertrophic chondrocytes in epiphyseal cartilage.

We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7 day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development.

Hydrogel-beta-TCP scaffolds and stem cells for tissue engineering bone.

Trabecular bone is a material of choice for reconstruction after trauma and tumor resection and for correction of congenital defects. Autologous bone grafts are available in limited shapes and sizes; significant donor site morbidity is another major disadvantage to this approach. To overcome these limitations, we used a tissue engineering approach to create bone replacements in vitro, combining bone-marrow-derived differentiated mesenchymal stem cells (MSCs) suspended in hydrogels and 3-dimensionally printed (3DP) porous scaffolds made of beta-tricalcium-phosphate (beta-TCP). The scaffolds provided support for the formation of bone tissue in collagen I, fibrin, alginate, and pluronic F127 hydrogels during culturing in oscillating and rotating dynamic conditions. Histological evaluation including toluidine blue, alkaline phosphatase, and von Kossa staining was done at 1, 2, 4, and 6 weeks. Radiographic evaluation and high-resolution volumetric CT (VCT) scanning, expression of bone-specific genes and biomechanical compression testing were performed at 6 weeks. Both culture conditions resulted in similar bone tissue formation. Histologically collagen I and fibrin hydrogels specimens had superior bone tissue, although radiopacities were detected only in collagen I samples. VCT scan revealed density values in all but the Pluronic F127 samples, with Houndsfield unit values comparable to native bone in collagen I and fibrin glue samples. Expression of bone-specific genes was significantly higher in the collagen I samples. Pluronic F127 hydrogel did not support formation of bone tissue. All samples cultured in dynamic oscillating conditions had slightly higher mechanical strength than under rotating conditions. Bone tissue can be successfully formed in vitro using constructs comprised of collagen I hydrogel, MSCs, and porous beta-TCP scaffolds.

Rough endoplasmic reticulum abnormalities in a patient with spondyloepimetaphyseal dysplasia with scoliosis, joint laxity, and finger deformities.

Iliac crest growth cartilage biopsy in spondyloepimetaphyseal dysplasia (SEMD) showed an endoplasmic reticulum storage disorder of epiphyseal and physeal chondrocytes. Biochemical analyses of iliac crest cartilage extracellular matrix showed no signs of deficits in any of the structural collagens types II, IX, or XI. The physis was abnormal by light microscopy with chondrocyte columnation replaced by clone-like cell accumulations surrounded by widened acellular cartilage septae. The rough endoplasmic reticulum (RER) of most chondrocytes was dilated. In some cells the RER contained homogeneous material but in most there were abnormal electron-dense accumulations. In some the material was seen in small amounts adjacent to the edge of the RER. In others, increasingly large amounts were seen that were randomly oriented and diffusely marginated. In many cells, assembly had progressed to well-marginated collections of wavy rod-like structures with a circular orientation parallel to the outer edges of the RER. The electron-dense accumulations measured from 34 to 40 nm in diameter. Mutations have prevented normal processing of collagen such that exit from the RER is abnormally slowed and abnormal self-assembly occurs within the dilated cisternae.