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. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.

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. At 25 weeks of age, the animals were euthanized and their proximal femurs were fixed and decalcified. Samples were sectioned and analyzed by histology to define extracellular matrix (ECM) structure, immunohistochemistry (IHC) to identify types II and X collagen, and histomorphometry to assess articular cartilage mean total and localized height and cell density. Statistics included nested mixed-effects ANOVA with p ≤ 0.05 considered statistically significant. RESULTS: Compared to controls, hypothyroid articular cartilage demonstrated statistically significant quantitative differences in mean tissue height, mean cell density and type II collagen localized zone height. Qualitative differences in ECM proteoglycans and overall collagen types were also found. Type X collagen was not detected in either hypothyroid or control articular cartilage specimens. CONCLUSIONS: Significant changes in articular cartilage structure in hypothyroid compared to control immature miniature swine suggest that thyroid hormone is critical in the growth and development of articular cartilage. CLINICAL SIGNIFICANCE: Understanding articular cartilage development in immature animal models may provide insight into healing or repair of degenerative human articular cartilage.

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. Specimens were then harvested and assessed by histological and gene expression analyses. Compared to controls, the presence of new bone formation and resorption in periosteum-allograft constructs was indicated in both histology and gene expression results over 40 weeks of implantation. Of several genes also examined for the first time, RANKL and SOST expression levels increased in a statistically significant manner, data suggesting that bone formation and the presence of increasing numbers of osteocytes in bone matrices had increased with time. The tissue-engineering strategy described in this study provides a possible means of improving delayed-union or non-union at the healing sites of segmental bone defects or bone fractures. The potential of periosteum and its resident cells could thereby be utilized effectively in tissue-engineering methods and tissue regenerative medicine.

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. Although genomic and protein data at high concentrations area GRGDS (71-83 pmol/cm(2)):BMP-2 (25 pmol/cm(2)) reveal an implicit acceleration on the hMSC differentiation timeline relative to the individual peptide concentrations, most of the GRGDS and BMP-2 combinations displayed significant antagonistic behavior during the hMSC differentiation. These data highlight the utility of the orthogonal gradient approach to aid in identifying optimal concentration ranges of translationally relevant peptides and growth factors for targeting cell lineage commitment.

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. While mitochondria protect cytosolic enzyme systems through this buffering capacity, the accumulation of calcium ions by mitochondria promotes the activity of enzymes of the tricarboxylic acid (TCA/Krebs) cycle, increases oxidative phosphorylation and ATP synthesis, and leads to changes in intramitochondrial pH. These pH alterations influence ion solubility and possibly the transitions and composition in the mineral phase structure of the granules. Based on these considerations, mitochondria are proposed to support the mineralization process by providing a mobile store of calcium and phosphate ions, in smaller cluster or larger granule form, while maintaining critical cellular activities. The rise in the mitochondrial calcium level also increases the generation of citrate and other TCA cycle intermediates that contribute to cell function and the development of extracellular mineral. This paper suggests that another key role of the mitochondrion, along with the effects just noted, is to supply phosphate ions, derived from the breakdown of ATP, to endolysosomes and autophagic vesicles originating in the endoplasmic reticulum and Golgi and at the plasma membrane. These many separate but interdependent mitochondrial functions emphasize the critical importance of this organelle in the cellular control of vertebrate mineralization.

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). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.

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 concept is fundamental to understanding both normal and abnormal mineralization, and it is reviewed in this article. Given this background, avenues for additional research studies in vertebrate mineralization will also be described. The latter include, for instance, how mineralization events subsequent to nucleation, that is, crystal growth and development, occur and whether they, too, are directed by collagen stereochemical parameters; whether mineralization can be expected in all spaces between collagen molecules; whether the side chains of charged amino acid residues actually point toward and into the hole and overlap collagen spaces to provide putative binding sites for calcium and phosphate ions; and what phenomena may be responsible for mineralization beyond hole and overlap zones and into extracellular tissue regions between collagen structural units. These questions will be discussed to provide a broader understanding of collagen contributions to potential mechanisms of vertebrate mineralization.

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. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≥ cranial ≥ mandibular; type II collagen, radial > ilium > cranial ≥ mandibular; runx2, cranial >>> radial > mandibular ≥ ilium; and BSP, ilium ≥ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.

Deposition of apatite in mineralizing vertebrate extracellular matrices: A model of possible nucleation sites on type I collagen.

The positions of charged residues in the primary sequence of amino acids comprising the molecular model of type I collagen, the major extracellular protein found in vertebrate tissues, have been earlier characterized by Chapman and Hardcastle [Chapman, J.A., and Hardcastle, R.A. (1974). The staining pattern of collagen fibrils. II. A comparison with patterns computer-generated from the amino acid sequence. Connect. Tissue Res. 2:151-159]. When the sequence of residues is packed in the quarter-staggered arrangement described originally by Hodge and Petruska [Hodge, A.J., and Petruska, J.A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In Aspects of Protein Structure, G.N. Ramachandran (ed.) pp. 289-300. New York: Academic Press] in two dimensions and in the quasi-hexagonal model of microfibrillar assembly and molecular packing structure in three dimensions detailed recently by Orgel et al. (Orgel, J.P.R.O., Miller, A., Irving, T.C., Fischetti, R.F., Hammersley, A.P., and Wess, T.J. (2001). The in situ supermolecular structure of type I collagen. Structure 9:1061-1069; Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci. U.S.A. 103: 9001-9005], the common sites of charged amino acids, specifically glutamic and aspartic acid, lysine and arginine, and hydroxylysine and histidine, of type I collagen have been examined in the present study and their locations determined in relation to one another. The respective positions of these amino acid residues are notable in several features in two dimensions within a single collagen triple helix as well as in adjacent helices. There are, first, numerous sites in which the same amino acid is adjacent in each of the three collagen helices. Second, many sites exist in which two of the same amino acids and one of the same charge are adjacent in the three helices. Third, the same two or three glutamic and/or aspartic amino acids are found in close proximity to amino acids with their counterparts, aspartic and glutamic acid, respectively. Fourth, several sites occur in which the same two or three amino acids of one charge are present in close proximity to the same two or three amino acids of opposite charge (glutamic acid and lysine or arginine residues or aspartic acid and lysine or arginine residues). Fifth, there are several sites where hydroxylysine contributes charged groups in place of one of the three lysine or arginine residues common in adjacent collagen helices. The strikingly repetitive and close nature of these specific charged groups in two dimensions is even more apparent when the molecular packing structure is investigated in three dimensions. In this instance, the most recent model of Orgel et al. [Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci. U.S.A. 103: 9001-9005] has been correlated for the first time with the model of Landis et al. [Landis, W.J., Song, M.J., Leith, A., McEwen, L., and McEwen, B. (1993). Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high voltage electron microscopic tomography and graphic image reconstruction. J. Struct. Biol. 110: 39-54] showing channels traversing molecular arrays of collagen. Here, many of the charged amino acid sites correspond to the known type I collagen hole zones defined by Hodge and Petruska [Hodge, A.J., and Petruska, J.A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In Aspects of Protein Structure, G.N. Ramachandran (ed.) pp. 289-300. New York: Academic Press]. As such, these residues present the locations highly likely to bind Ca(2+) and [Formula: see text] ions in stereochemical configurations that could serve directly as nucleation centers for the subsequent growth and development of apatite crystals representing initial events in vertebrate mineralization. Based on these results, type I collagen appears to provide a molecular framework for direct formation of apatite without the necessary intervention or mediation of other molecules in extracellular matrices of vertebrate calcifying tissues.

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. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

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

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 Ca10(PO4)6(OH)2) 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. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.

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. Neocartilages from normal and microtia surgical tissues were found equivalent in their dimensions, qualitative degree of proteoglycan and elastic fiber staining, and quantitative gene expression levels of types II and III collagen, elastin, and SOX5. Microarray analysis, applied for the first time for normal and microtia neocartilage comparison, yielded no genes that were statistically significantly different in expression between these two sample groups. These results support use of microtia tissue as a cell source for normal auricular reconstruction. Comparison of normal and microtia cells, each seeded on nanoPGA and supplemented with BMP7 in a slow-release hydrogel, showed statistically significant differences in certain genes identified by microarray analysis. Such differences were also noted in several analyses comparing counterpart seeded cells without BMP7. Summary data suggest a possible application for BMP7 in microtia cartilage regeneration and encourage further studies to elucidate whether such genotypic differences translate to phenotypic characteristics of the human microtic ear. The present work advances understanding relevant to the potential clinical use of microtia surgical remnants as a suitable cell source for tissue engineering of the pinna.

Mineral deposition in the extracellular matrices of vertebrate tissues: identification of possible apatite nucleation sites on type I collagen.

The possible means by which type I collagen may mediate mineralization in normal vertebrate bone, tendon, dentin and cementum as well as in pathological mineral formation are not fully understood. One consideration in this regard is that the structure of the protein is somehow important in binding calcium and phosphate ions in a stereochemical configuration conducive to nucleation of apatite crystals. In the present study, type I collagen, packed in a quarter-staggered arrangement in two dimensions and a quasi-hexagonal model of microfibrillar assembly in three dimensions, has been examined in terms of several of its charged amino acid residues. These included glutamic and aspartic acid, lysine, arginine, hydroxylysine and histidine, whose positions along the three alpha-chain axes of the collagen molecule were determined with respect to each other. It was found that the locations of these residues specified sites uniquely suited as potential apatite nucleation centers following binding of calcium and phosphate ions. From this analysis, it would appear that type I collagen provides a template of charged amino acid residues that dictates ion binding critical to subsequent nucleation events for mineral formation in vertebrate tissues.

Tissue engineering models of human digits: effect of periosteum on growth plate cartilage development.

Tissue-engineered middle phalanx constructs of human digits were investigated to determine whether periosteum wrapped partly about model midshafts mediated cartilage growth plate formation. Models were fabricated by suturing ends of polymer midshafts in a human middle phalanx shape with polymer sheets seeded with heterogeneous chondrocyte populations from bovine articular cartilage. Half of each midshaft length was wrapped with bovine periosteum. Constructs were cultured, implanted in nude mice for up to 20 weeks, harvested and treated histologically to assess morphology and cartilage proteoglycans. After 20 weeks of implantation, chondrocyte-seeded sheets adjacent to periosteum-wrapped midshaft halves established cartilage growth plates resembling normal tissue in vivo. Sheets adjacent to midshafts without periosteum had disorganized cells and no plate formation. Proteoglycans were present at both midshaft ends. Periosteum appears to guide chondrocytes toward growth plate cartilage organization and tissue engineering provides means for carefully examining construct development of this tissue.

Histochemical analyses of tissue-engineered human menisci.

The field of tissue engineering remains one of the least explored areas of current meniscal research but holds great promise. In this investigation, meniscal fibrochondrocytes were isolated from fresh human meniscal tissue and seeded onto synthetic polyglycolic acid (PGA) scaffolds. Constructs were implanted into the dorsal subcutaneous space of athymic nude mice. Control scaffolds, devoid of meniscal cells, were simultaneously implanted in additional mice. Constructs were harvested over 12 weeks and treated with a variety of histochemical stains to analyze general specimen morphology, cellular viability and proliferation, and collagen secretion. Results indicate that meniscal fibrochondrocyte proliferation increased over the time of implantation with cellular consolidation occurring as the PGA scaffolding was progressively hydrolyzed. Collagen production also increased over time. There were favorable similarities between constructs and human meniscal controls in terms of cellular morphology, phenotypic expression, and collagen production. These initial findings demonstrate procedures supporting proliferation of meniscal fibrochondrocytes, expression of fibrochondral phenotype, and the formation of putative meniscal tissue.

Tissue engineering a model for the human ear: assessment of size, shape, morphology, and gene expression following seeding of different chondrocytes.

This study examines the tissue engineering of a human ear model through use of bovine chondrocytes isolated from four different cartilaginous sites (nasoseptal, articular, costal, and auricular) and seeded onto biodegradable poly(l-lactic acid) and poly(L-lactide-epsilon-caprolactone) (50 : 50) polymer ear-shaped scaffolds. After implantation in athymic mice for up to 40 weeks, cell/scaffold constructs were harvested and analyzed in terms of size, shape, histology, and gene expression. Gross morphology revealed that all the tissue-engineered cartilages retained the initial human auricular shape through 40 weeks of implantation. Scaffolds alone lost significant size and shape over the same period. Quantitative reverse transcription-polymerase chain reaction demonstrated that the engineered chondrocyte/scaffolds yielded unique expression patterns for type II collagen, aggrecan, and bone sialoprotein mRNA. Histological analysis showed type II collagen and proteoglycan to be the predominant extracellular matrix components of the various constructs sampled at different implantation times. Elastin was also present but it was found only in constructs seeded with auricular chondrocytes. By 40 weeks of implantation, tissue-engineered cartilage of costal origin became calcified, marked by a notably high relative gene expression level of bone sialoprotein and the presence of rigid, nodular protrusions formed by mineralizing rudimentary cartilaginous growth plates. The collective data suggest that nasoseptal, articular, and auricular cartilages represent harvest sites suitable for development of tissue-engineered human ear models with retention over time of three-dimensional construct architecture, gene expression, and extracellular matrix composition comparable to normal, nonmineralizing cartilages. Calcification of constructs of costal chondrocyte origin clearly shows that chondrocytes from different tissue sources are not identical and retain distinct characteristics and that these specific cells are inappropriate for use in engineering a flexible ear model.

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. Quantitative polymerase chain reaction analysis demonstrates that mRNA expression of the non-collagenous genes of bone sialoprotein, osteopontin, and osteocalcin corresponds well with the temporal and spatial onset and progression of mineralization. Immunolocalization separately confirms the synthesis and secretion of these matrix molecules. The expression of other non-collagenous genes such as decorin does not show strong correlation with turkey leg tendon mineralization, and expression of vimentin, a cytoskeletal component which may be regulated by biomechanical factors in the tendon, may lead to inhibition of osteocalcin expression during the development and mineralization of the tissue. The overall results of this work provide insight into direct temporal and spatial relations between the genes and proteins of interest as well as the formation and deposition of mineral in the avian tendon model.

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. Thirteen female New Zealand white rabbits (five 15-week-old and eight 19-week-old animals) were euthanized and physes harvested from their proximal and distal femurs and proximal tibiae. Harvested physes were divided into groups for histological, immunohistochemical (IHC), and reverse transcription-quantitative polymerase chain reaction analyses. All physes analyzed demonstrated no apparent differences in morphology or proteoglycan staining intensity on histological examination or in type II collagen presence determined by IHC study. Histomorphometric measures of physeal height as well as gene expression of type II collagen and aggrecan were found to be statistically significantly equivalent (p < 0.05) among the three different bones from the total number of rabbits. Summary data suggest that the structural similarities and statistical equivalence determined among the various physes investigated in the rabbit validate these tissues in this species for use as surrogate controls by which physeal abnormalities may be compared and characterized in the absence of otherwise normal control tissues. Other species may exhibit the same similarities and equivalence among different physes so that such tissues may serve in like manner as controls for assessing a variety of orthopedic conditions, including those occurring in humans.

Evaluation of bioreactor-cultivated bone by magnetic resonance microscopy and FTIR microspectroscopy.

We present a three-dimensional mineralizing model based on a hollow fiber bioreactor (HFBR) inoculated with primary osteoblasts isolated from embryonic chick calvaria. Using non-invasive magnetic resonance microscopy (MRM), the growth and development of the mineralized tissue around the individual fibers were monitored over a period of 9 weeks. Spatial maps of the water proton MRM properties of the intact tissue, with 78 microm resolution, were used to determine changes in tissue composition with development. Unique changes in the mineral and collagen content of the tissue were detected with high specificity by proton density (PD) and magnetization transfer ratio (MTR) maps, respectively. At the end of the growth period, the presence of a bone-like tissue was verified by histology and the formation of poorly crystalline apatite was verified by selected area electron diffraction and electron probe X-ray microanalysis. FTIR microspectroscopy confirmed the heterogeneous nature of the bone-like tissue formed. FTIR-derived phosphate maps confirmed that those locations with the lowest PD values contained the most mineral, and FTIR-derived collagen maps confirmed that bright pixels on MTR maps corresponded to regions of high collagen content. In conclusion, the spatial mapping of tissue constituents by FTIR microspectroscopy corroborated the findings of non-invasive MRM measurements and supported the role of MRM in monitoring the bone formation process in vitro.

Type XXVII collagen at the transition of cartilage to bone during skeletogenesis.

COL27A1 is a member of the collagen fibrillar gene family and is expressed in cartilaginous tissues including the anlage of endochondral bone. To begin to understand its role in skeletogenesis, the temporospatial distributions of its RNA message and protein product, type XXVII collagen, were determined in developing human skeletal tissues. Laser capture microdissection and quantitative reverse-transcription polymerase chain reaction demonstrated that gene expression occurred throughout the growth plate and that it was higher in the resting and proliferative zones than in hypertrophic cartilage. Immunohistochemical analyses showed that type XXVII collagen was most evident in hypertrophic cartilage at the primary ossification center and at the growth plate and that it accumulated in the pericellular matrix. Synthesis of type XXVII collagen overlapped partly with that of type X collagen, a marker of chondrocyte hypertrophy, preceded the transition of cartilage to bone, and was associated with cartilage calcification. Immunogold electron microscopy of extracted ECM components from mouse growth plate showed that type XXVII collagen was a component of long non-banded fibrous structures, filamentous networks, and thin banded fibrils. The timing and location of synthesis suggest that type XXVII collagen plays a role during the calcification of cartilage and the transition of cartilage to bone.