Effects of mineralization on the hierarchical organization of collagen-a synchrotron X-ray scattering and polarized second harmonic generation study.

The process of mineralization fundamentally alters collagenous tissue biomechanics. While the structure and organization of mineral particles have been widely studied, the impact of mineralization on collagen matrix structure, particularly at the molecular scale, requires further investigation. In this study, synchrotron X-ray scattering (XRD) and polarization-resolved second harmonic generation microscopy (pSHG) were used to study normally mineralizing turkey leg tendon in tissue zones representing different stages of mineralization.

Toward understanding the cellular control of vertebrate mineralization: The potential role of mitochondria.

This review examines the possible role of mitochondria in maintaining calcium and phosphate ion homeostasis and participating in the mineralization of bone, cartilage and other vertebrate hard tissues. The paper builds on the known structural features of mitochondria and the documented observations in these tissues that the organelles contain calcium phosphate granules. Such deposits in mitochondria putatively form to buffer excessively high cytosolic calcium ion concentrations and prevent metabolic deficits and even cell death.

Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs.

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction.

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues .

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca(PO)(OH)) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue.

Induced hypothyroidism alters articular cartilage in skeletally immature miniature swine.

Purpose/aim: Thyroid hormone has been implicated in the normal growth and development of articular cartilage; however, its effect on a disease state, such as hypothyroidism, is unknown. The purpose of this investigation was to compare normal articular cartilage from proximal femurs of immature miniature swine to proximal femurs from hypothyroid-induced immature miniature swine.

Materials And Methods: Two 11-week-old male Sinclair miniature swine were made hypothyroid by administration of 6-propyl-2-thiouracil (PTU) in their drinking water; two control animals did not receive PTU.

Characterization of Tissue-Engineered Human Periosteum and Allograft Bone Constructs: The Potential of Periosteum in Bone Regenerative Medicine.

Delayed-union or non-union between a host bone and a graft is problematic in clinical treatment of segmental bone defects in orthopedic cases. Based on a preliminary study of human periosteum allografts from this laboratory, the present work has extensively investigated the use of human cadaveric tissue-engineered periosteum-allograft constructs as an approach to healing such serious orthopedic surgical situations. In this current report, human cadaveric periosteum-wrapped bone allografts and counterpart controls without periosteum were implanted subcutaneously in athymic mice (nu/nu) for 10, 20, and, for the first time, 40 weeks.

An analytical study of neocartilage from microtia and otoplasty surgical remnants: A possible application for BMP7 in microtia development and regeneration.

To investigate auricular reconstruction by tissue engineering means, this study compared cartilage regenerated from human chondrocytes obtained from either microtia or normal (conchal) tissues discarded from otoplasties. Isolated cells were expanded in vitro, seeded onto nanopolyglycolic acid (nanoPGA) sheets with or without addition of bone morphogenetic protein-7 (BMP7), and implanted in nude mice for 10 weeks. On specimen harvest, cartilage development was assessed by gross morphology, histology, and RT-qPCR and microarray analyses.

Three-dimensional structural interrelations between cells, extracellular matrix, and mineral in normally mineralizing avian leg tendon.

The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents.

An investigation to validate the equivalence of physes obtained from different anatomic regions in a single animal species: Implications for choosing experimental controls in clinical studies.

Control tissue in studies of various orthopedic pathologies is difficult to obtain and presumably equivalent biopsies from other anatomic sites have been utilized in its place. However, for growth plates, different anatomic regions are subject to dissimilar mechanical forces and produce disproportionate longitudinal growth. The purpose of this study was to compare gene expression and structure in normal physes from different anatomic regions within a single animal species to determine whether such physes were equivalent.

Correlations between gene expression and mineralization in the avian leg tendon.

Certain avian tendons have been studied previously as a model system for normal mineralization of vertebrates in general. In this regard, the gastrocnemius tendon in the legs of turkeys mineralizes in a well defined temporal and spatial manner such that changes in the initial and subsequent events of mineral formation can be associated with time and specific locations in the tissue. In the present investigation, these parameters and mineral deposition have been correlated with the expression of several genes and the synthesis and secretion of their related extracellular matrix proteins by the composite tenocytes of the tendon.

A Method for the Immunohistochemical Identification and Localization of Osterix in Periosteum-Wrapped Constructs for Tissue Engineering of Bone.

A novel immunohistochemistry (IHC) approach has been developed to label and localize osterix, a bone-specific transcription factor, within formalin-fixed, paraffin-embedded, tissue-engineered constructs uniquely containing synthetic polymers and human periosteal tissue. Generally, such specimens consisting in part of polymeric materials and mineral are particularly difficult for IHC identification of proteins. Samples here were fabricated from human periosteum, electrospun poly-l-lactic acid (PLLA) nanofibers, and polycaprolactone/poly-l-lactic acid (PCL/PLLA, 75/25) scaffolds and harvested following 10 weeks of implantation in athymic mice.

Long-Term Comparison between Human Normal Conchal and Microtia Chondrocytes Regenerated by Tissue Engineering on Nanofiber Polyglycolic Acid Scaffolds.

Background: Previous regeneration studies of auricle-shaped cartilage by tissue engineering leave unresolved whether the chondrocyte phenotype from human auricular chondrocytes seeded onto polymeric scaffolds is retained over the long term and whether microtia remnants may be a viable cell source for auricular reconstruction.

Methods: Chondrocytes were isolated from human ears, either normal conchal ear or microtia cartilage remnants, expanded in vitro, and seeded onto nanoscale-diameter polyglycolic acid sheets. These tissue-engineered constructs were implanted into athymic mice for up to 40 weeks.

Recent Applications of Coaxial and Emulsion Electrospinning Methods in the Field of Tissue Engineering.

Electrospinning has emerged as an effective method of producing nanoscale fibers for use in multiple fields of study. One area of significant interest is nanofiber utilization for tissue engineering because the nanofibrous mats can mimic the native extracellular matrix of biological tissues. A logical next step is the inclusion of certain molecules and compounds to accelerate or increase the efficacy of tissue regeneration.

Microarray analysis of slipped capital femoral epiphysis growth plates.

Background: Microarray technology has been used to analyze gene expression in patients with and without slipped capital femoral epiphysis (SCFE).

Methods: Proximal femoral physis core biopsies from two patients with SCFE were compared with two control specimens from age-matched patients without SCFE. Extracted RNA from frozen ground samples was subjected to microarray analysis with data tests for statistical significance between SCFE and control tissues.

Concentration-Dependent hMSC Differentiation on Orthogonal Concentration Gradients of GRGDS and BMP-2 Peptides.

Self-assembled monolayer substrates containing tethered orthogonal concentration profiles of GRGDS (glycine/arginine/glycine/aspartic acid/serine) and BMP-2 (bone morphogenetic protein) peptides are shown to accelerate or decelerate, depending on the concentrations, the proliferation and osteoblastic differentiation of human mesenchymal stem cell (hMSC) populations in vitro without the use of osteogenic additives in culture medium. Concurrently, the single peptide gradient controls (GRGDS or BMP-2 only) induce significantly different proliferation and differentiation behavior from the orthogonal substrates. Bone sialoprotein (BSP) and Runt-related transcription factor 2 (Runx2) PCR data acquired from hMSC populations isolated by laser capture microdissection correspond spatially and temporally to protein marker data obtained from immunofluorescent imaging tracking of the differentiation process.

Matrix vesicles: Are they anchored exosomes?

Numerous studies have documented that matrix vesicles are unique extracellular membrane-bound microparticles that serve as initial sites for mineral formation in the growth plate and most other vertebrate mineralizing tissues. Microparticle generation is not confined to hard tissues, as cells in soft tissues generate similar structures; numerous studies have shown that a common type of extracellular particle, termed an exosome, a product of the endosomal pathway, shares many characteristics of matrix vesicles. Indeed, analyses of size, morphology and lipid and protein content indicate that matrix vesicles and exosomes are homologous structures.

Gene expression differences between ruptured anterior cruciate ligaments in young male and female subjects.

Background: The incidence of anterior cruciate ligament (ACL) injuries is two to eightfold greater in female compared with male athletes. Anatomic, hormonal, and neuromuscular factors have been associated with this disparity. This study compared gene expression and structural features in ruptured but otherwise normal ACL tissue from young female and male athletes.

Molecular mechanisms for intrafibrillar collagen mineralization in skeletal tissues.

The critical role of the self-assembled structure of collagen in skeletal mineralization is long recognized, yet the angstrom to tens of nanometers length-scale nucleation mechanism of calcium phosphate mineral (Ca-Pi) remains unclear. Here, by constructing three-dimensional structure of collagen fibril, we report direct computational evidence of intrafibrillar Ca-Pi nucleation in the collagen matrix and illustrate the crucial role of charged amino acid sidechains of collagen molecules in nucleation. The all-atom Hamiltonian replica exchange molecular dynamics simulation shows that these charged sidechains are oriented toward the fibril "hole zones" and significantly template nucleation with amorphous Ca-Pi phase, ∼1.

Refinement of collagen-mineral interaction: a possible role for osteocalcin in apatite crystal nucleation, growth and development.

Mineralization of vertebrate tissues such as bone, dentin, cementum, and calcifying tendon involves type I collagen, which has been proposed as a template for calcium and phosphate ion binding and subsequent nucleation of apatite crystals. Type I collagen thereby has been suggested to be responsible for the deposition of apatite mineral without the need for non-collagenous proteins or other extracellular matrix molecules. Based on studies in vitro, non-collagenous proteins, including osteocalcin and bone sialoprotein, are thought to mediate vertebrate mineralization associated with type I collagen.

Effects of FGF-2 and OP-1 in vitro on donor source cartilage for auricular reconstruction tissue engineering.

Objective: Microtia is a congenital partial or total loss of the external ear with current treatment approaches involving autologous construction from costal cartilage. Alternatively, tissue engineering provides possible use of normal or microtia auricular chondrocytes harvested from patients. This study investigated effects in vitro of basic fibroblast growth factor (FGF-2) and osteogenic protein 1 (OP-1) on human pediatric normal and microtia auricular chondrocytes and their potential proliferation and differentiation for cellular expansion.

The effects of hypothyroidism on the proximal femoral physis in miniature swine.

As a potential means of comparing hypothyroidism in humans, this work intended to establish a defined hypothyroid state in immature miniature swine and evaluate specific molecular, cellular, and extracellular responses of their growth plates. Two male, 11-week-old Sinclair miniature swine were given 6-propyl-2-thiouracil (PTU) in their water and two other like animals (controls) were provided water without PTU. Blood levels of thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxin (T4) were monitored weekly.

Association of calcium and phosphate ions with collagen in the mineralization of vertebrate tissues.

Among the vertebrate species, collagen is the most abundant protein and is associated with mineralization of their skeleton and dentition in all tissues except enamel. In such tissues, bones, calcifying tendon, dentin, and cementum are comprised principally of type I collagen, which has been proposed as a template for apatite mineral formation. Recent considerations of the interaction between type I collagen and calcium and phosphate ions as the major constituents of apatite have suggested that collagen polypeptide stereochemistry underlies binding of these ions at sites within collagen hole and overlap regions and leads to nucleation of crystals.

The effects of GATA-1 and NF-E2 deficiency on bone biomechanical, biochemical, and mineral properties.

Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase).

A study in vivo of the effects of a static compressive load on the proximal tibial physis in rabbits.

Background: The effect of compression on the physis is generally defined by the Hueter-Volkmann principle, in which decreased linear growth of the physis results from increased compression. This investigation examined whether mechanically induced compression of rabbit physes causes changes in gene expression, cells, and extracellular components that promote physeal resilience and strength (type-II collagen and aggrecan) and cartilage hypertrophy (type-X collagen and matrix metalloprotease-13).

Methods: Static compressive loads (10 N or 30 N) were applied for two or six weeks across one hind limb proximal tibial physis of thirteen-week-old female New Zealand White rabbits (n = 18).

The nature and role of periosteum in bone and cartilage regeneration.

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels.