On how nucleus-endplate integration is achieved at the fibrillar level in the ovine lumbar disc.

The intervertebral disc nucleus has traditionally been viewed as a largely unstructured amorphous gel having little obvious integration with the cartilaginous endplates (CEPs). However, recent work by the present authors has provided clear evidence of structural cohesion across the nucleus-endplate junction via a distinctive microanatomical feature termed insertion nodes. The aim of this study was to explore the nature of these insertion nodes at the fibrillar level. Specially prepared vertebra-nucleus-vertebra composite samples from ovine lumbar motion segments were extended axially and chemically fixed in this stretched state, and then decalcified. Sections taken from the samples were prepared for examination by scanning electron microscopy. A close morphological correlation was obtained between previously published optical microscopic images of the nodes and those seen using low magnification SEM. Progressively high magnifications provided insight into the fibrillar-level modes of structural integration across the nucleus-endplate junction. The closely packed fibrils of the CEP were largely parallel to the vertebral endplate and formed a dense, multi-layer substrate within which the nodal fibrils appeared to be anchored. Our idealised structural model proposes a mechanism by which this integration is achieved. The nodal fibrils, in curving into the CEP, are locked in place within its close-packed layers of transversely aligned fibrils, and probably at multiple levels. Secondly, there appears to be a subtle interweaving of the strongly aligned nodal fibrils with the multi-directional endplate fibrils. It is suggested that this structural integration provides the nucleus with a form of tethered mobility that supports physiological functions quite distinct from the primary strength requirements of the disc.

Local anisotropy in mineralized fibrocartilage and subchondral bone beneath the tendon-bone interface.

The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.

Collagen morphology in human meniscal attachments: a SEM study.

Qualitative analysis of meniscal attachments from five human knees was completed using scanning electron microscopy (SEM). In addition, quantitative analysis to determine the collagen crimping angle and length in each attachment was done. Morphological differences were revealed between the distinct zones of the attachments from the meniscus transition to the bony insertion. Collagen fibers near to the meniscus appeared inhomogeneous in a radial cross-section view. The sheath surrounding the fibers seemed loose compared with the membrane wrapping around the fibers in the menisci. The midsubstance of human meniscal attachments was composed of collagen fibers running parallel to the longitudinal axis, with a few fibers running obliquely, and others transversely. The bony insertion showed that the crimping pattern vanishes as the collagen fibers approach the fibrocartilagenous enthesis. There were no differences between attachments for crimping angle or length. Collagen crimping angles for all attachments were similar with values of approximately 22°. Crimp length values tended to be smaller for the medial attachments (MA: 4.76 ± 1.95 µm; MP: 3.72 ± 2.31 µm) and higher for the lateral (LA: 6.49 ± 2.34 µm, LP: 6.91 ± 2.29 µm). SEM was demonstrated to be an effective method for revealing the morphology of fibrous connective tissue. The data of collagen fiber length and angle found in this study will allow for better development of microstructural models of meniscal attachments. This study will help to better understand the relation between the morphology and the architecture of collagen and the mechanical behavior of meniscal attachments.

Facilitated tendon-bone healing by local delivery of recombinant hepatocyte growth factor in rabbits.

PURPOSE: This study was performed to evaluate the therapeutic effect of hepatocyte growth factor (HGF) on tendon-bone healing in a rabbit model. METHODS: In adult rabbits the long digital extensor tendon was detached from the lateral femoral condyle, and the free end of the tendon was inserted into a tunnel drilled into the proximal tibial metaphysis. Cancellous bone obtained during drilling of the tibial hole was soaked in saline solution or solution containing 100-microg/mL human recombinant HGF and then transplanted into the bone tunnel. Junctional healing between the tendon and the bone was evaluated by histologic analysis and uniaxial load-to-failure testing at 2, 4, 6, 8, and 12 weeks after surgery. RESULTS: In the saline solution-treated control group, Sharpey-like fibers, which connected the tendon graft and the bone tissue, appeared 6 weeks after treatment. At 8 weeks after treatment, maturation of lamellar bone was seen, and at 12 weeks, the adhesion between tendon and bone appeared to be supported by indirect insertion of fibrocartilaginous tissue, wherein the border between the fibrocartilaginous tissue and tendon or bone was significant. In the HGF-treated group, the fibrous tissues were parallel to the load axis, and lamellar bone and Sharpey-like fibers appeared as early as 4 weeks after treatment. At 12 weeks, junctional tissue, characterized by a continuous 4-layer structure of bone, calcified cartilage, fibrocartilage, and tendon, was regenerated by a direct insertion. On biomechanical testing, the HGF-treated group had significantly better biomechanical properties than the control group at 2 and 4 weeks. The histologic improvement caused by HGF treatment was associated with the biomechanical improvement. CONCLUSIONS: Local administration of recombinant HGF promotes the adhesive healing process at the tendon-bone junction, both histologically and mechanically, after ligament reconstruction in a rabbit model. CLINICAL RELEVANCE: Application of HGF may be considered as a new therapeutic approach to accelerate healing and rehabilitation after ligament reconstruction.

The elastic fibre network of the human lumbar anulus fibrosus: architecture, mechanical function and potential role in the progression of intervertebral disc degeneration.

Elastic fibres are critical constituents of dynamic biological structures that functionally require elasticity and resilience. The network of elastic fibres in the anulus fibrosus of the intervertebral disc is extensive, however until recently, the majority of histological, biochemical and biomechanical studies have focussed on the roles of other extracellular matrix constituents such as collagens and proteoglycans. The resulting lack of detailed descriptions of elastic fibre network architecture and mechanical function has limited understanding of the potentially important contribution made by elastic fibres to healthy disc function and their possible roles in the progression of disc degeneration. In addition, it has made it difficult to postulate what the consequences of elastic fibre related disorders would be for intervertebral disc behaviour, and to develop treatments accordingly. In this paper, we review recent and historical studies which have examined both the structure and the function of the human lumbar anulus fibrosus elastic fibre network, provide a synergistic discussion in an attempt to clarify its potentially critical contribution both to normal intervertebral disc behaviour and the processes relating to its degeneration, and recommend critical areas for future research.

Ultrastructural differences in cranial cruciate ligaments from dogs of two breeds with a differing predisposition to ligament degeneration and rupture.

Cranial (anterior) cruciate ligament (CCL) samples were obtained from dogs of the Labrador retriever (LR) and greyhound (GH) breeds, of which the former but not the latter is predisposed to CCL rupture. Electron microscopy revealed that the collagen fibril diameters of GHs were larger than those of LRs (P=0.03). Histological examination revealed a "fibrocartilaginous" appearance of CCLs in seven of eight GHs, and, to a lesser extent, in three of eight LRs. The formation of fibrocartilage is clearly not a disadvantage to the healthy racing GH, and cannot be regarded as a pathological degeneration in this breed. It is suggested that fibrocartilage is formed as a beneficial physiological adaptation to the compression of CCLs caused by tensile stress as a result of the tightening of two twisted bands. Fibrocartilage would appear to protect CCLs in the GH, but it may be indicative of a mild degenerative change, which may eventually lead to rupture in the LR.

Does decalcification alter the tissue sound speed of rabbit supraspinatus tendon insertion? In vitro measurement using scanning acoustic microscopy.

Failure of the tendon or ligament insertions is one of the most common injuries in the Orthopaedic field. To elucidate the pathogenesis of those injuries, the authors had attempted to measure the tissue sound speed that could be correlated to its elasticity using scanning acoustic microscopy (SAM). For the application of SAM to tendon or ligament insertions, it was necessary to determine the role of decalcification in SAM measurements since mineralized tissues including bone or mineralized fibrocartilage were present at the insertion site. To assess whether decalcification alters the tissue sound speed or not, supraspinatus tendon insertion of six Japanese white rabbits were measured with SAM operating in the frequency range of 50-150 MHz. Right supraspinatus tendons attached to the humeral head were cut into two pieces at the center of the tendon. Then, they were fixed with 10% neutralized formalin for 12 h. In each specimen, medial half was not decalcified, while lateral half was decalcified with ethylene-diamine-tetra-acetic acid (EDTA). After embedding in paraffin, 5 microm thick specimens were prepared for the measurement using SAM. The mean sound speed in each histologic zone was evaluated, and subsequently compared to that measured in the undecalcified and the decalcified specimens. Mean sound speed of non-mineralized fibrocartilage was 1544 m/s in the undecalcified specimens, while the value of 1541 m/s was determined in the decalcified ones. On the other hand, it decreased 2-3% after decalcification in the mineralized tissue including mineralized fibrocartilage and bone (mineralized fibrocartilage: undecalcified = 1648 m/s, decalcified = 1604 m/s; bone: undecalcified = 1716 m/s, decalcified = 1677 m/s). However, no significant differences were found between the undecalcified and the decalcified specimens (non-mineralized fibrocartilage: p = 0.84, mineralized fibrocartilage: p = 0.35, bone: p = 0.28). These results indicate that SAM could be applied to determine the properties of the tendon or the ligament insertions after decalcification with EDTA. Although SAM is applicable only for in vitro experimental study, it is expected that these data will contribute to better understanding concerning the biomechanics of tendon or ligament insertions as well as the pathogenesis of their failure at a microscopic level.

Enhanced regeneration of the ligament-bone interface using a poly(L-lactide-co-ε-caprolactone) scaffold with local delivery of cells/BMP-2 using a heparin-based hydrogel.

Recently, the ligament-bone (LTB) junction has been emphasized for the effective transmission of mechanical force and the reduction in stress concentration between the soft ligament and hard bone tissue. The aim of this study was to regenerate an integrated LTB interface by inoculating LTB-relevant cells, isolated from fibrocartilage (FC) or ligament (LIG), separately into each designated region in a single porous cylindrical PLCL scaffold. An injectable, heparin-based hydrogel that has proved to be effective in the culture of chondrocytes as well as the sustained release of growth factor was employed to locally deliver fibrochondrocytes and osteoinductive bone morphogenetic protein-2 (BMP-2) into the FC region, to promote FC regeneration. In in vitro experiments the hydrogel-combined FC systems produced significantly larger amounts of calcium and glycosaminoglycans (GAGs), but less collagen and DNA than FC samples without the hydrogel and all LIG samples. After in vivo subcutaneous implantation in mice for 8 weeks the secreted calcium and GAG contents of the hydrogel-containing FC samples were superior or similar to those of the in vitro hydrogel-containing FC samples at 6 weeks. As a result of the enhanced production of calcium and GAG, the in vivo hydrogel-containing FC samples produced the highest compressive modulus among all samples. Histological and immunofluorescence analysis as well as elemental analysis also confirmed a denser and more homogeneous distribution of calcium, GAG, osteocalcin and neovascularization marker in the in vitro/in vivo hydrogel-containing FC systems than those without hydrogel. These results also show the beneficial effects of BMP-2 added using the hydrogel. In summary, the use of a heparin-based hydrogel for the local delivery of fibrochondrocytes and BMP-2 could accelerate the maturation and differentiation of LTB-specific FC tissues, and it was also possible to recreate the unique stratification of calcified FC and LIG tissues in a single porous PLCL scaffold in terms of both biochemical and biomechanical properties.

Murine metapodophalangeal sesamoid bones: morphology and potential means of mineralization underlying function.

Normal murine metapodophalangeal sesamoid bones, closely associated with tendons, were examined in terms of their structure and mineralization with reference to their potential function following crystal deposition. This study utilized radiography, whole mount staining, histology, and conventional electron microscopy to establish a maturation timeline of mineral formation in 1- to 6-week-old metapodophalangeal sesamoids from CD-1 mice. An intimate cellular and structural relationship was documented in more detail than previously described between the sesamoid bone, tendon, and fibrocartilage enthesis at the metapodophalangeal joint. Sesamoid calcification began in 1-week lateral sesamoids of the murine metacarpophalangeal joint of the second digit. All sesamoids were completely calcified by 4 weeks. Transmission electron microscopy of 2-week metacarpophalangeal sesamoids revealed extensive Type I collagen in the associated tendon and fibrocartilage insertion sites and Type II collagen and proteoglycan networks in the interior of the sesamoid. No extracellular matrix vesicles were documented. The results demonstrate that murine sesamoid bones consist of cartilage elaborated by chondrocytes that predominantly synthesize and secrete Type II collagen and proteoglycan. Type II collagen and proteoglycans appear responsible for the onset and progression of mineral formation in this tissue. These data contribute to new understanding of the biochemistry, ultrastructure, and mineralization of sesamoids in relation to other bones and calcifying cartilage and tendon of vertebrates. They also reflect on the potentially important but currently uncertain function of sesamoids as serving as a fulcrum point along a tendon, foreshortening its length and altering advantageously its biomechanical properties with respect to tendon-muscle interaction.

Micromass culture of human anulus cells: morphology and extracellular matrix production.

STUDY DESIGN: Micromass culture was assessed as a cell culture microenvironment for anulus cells from the human intervertebral disc. OBJECTIVE: To determine whether the micromass culture technique might be useful for the culture of human anulus cells. SUMMARY OF BACKGROUND DATA: Culture of cells in micromass has been traditionally used as a method to culture chondrocytes in a three-dimensional (3D) microenvironment with specialized chondrocyte media which allows expression of the chondrocytic phenotype. Recently it has also been used for disc cell 3D culture. METHODS: Following approval of our human subjects Institutional Review Board, cells isolated from human anulus intervertebral disc tissue was cultured in micromass culture under control conditions or with addition of 5 ng/mL transforming growth factor-beta (TGF-beta). Cultures were grown for 7 days, and then analyzed for morphology with light microscopy, for extracellular matrix (ECM) production with transmission electron microscopy and quantitative measurement of total sulfated proteoglycan production. Immunohistochemistry was also performed to assess types I and II collagen, decorin, keratan sulfate, and chondroitin sulfate content of ECM. RESULTS: Human anulus cells form multilayered colonies when cultured with minimal media and 20% fetal bovine serum in the micromass methodology. Stimulation of ECM production occurs when 5 ng/mL TGF-beta was added to the micromass media. TGF-beta also significantly increased the production of sulfated proteoglycans (P = 0.026). Under both control and TGF-beta-supplementation, the resulting micromass formed by anulus cells is not as compact as the micromass which results when stem cells cultured in chondrogenic media. Ultrastructural studies showed the presence of apoptotic cells and the presence of peroxisomes within cells. Immunohistochemical studies on production of type I collagen, decorin and keratan sulfate showed that there was localized production of these ECM components in focal regions; chondroitin sulfate and type II collagen, however, showed a more uniform overall production by cells within the micromass. CONCLUSION: Human anulus cells were successfully cultured under micromass conditions in nonchondrogenic media and with TGF-beta supplementation which increased ECM production. The resulting anulus cell micromass, however, was not as rounded or compact as that which occurs with routine chondrocyte micromass or stem cells induced into chondrocyte differentiation. The presence of peroxisomes noted on ultrastructural studies may reflect cell stress or uneven distribution of nutrition within the micromass during the 7-day micromass culture period. Immunohistochemical studies showed nonuniform ECM gene expression and production within the micromass, suggesting variable gene expression patterns with this culture method.

Different behavior of meniscal cells in collagen II/I,III and Hyaff-11 scaffolds in vitro.

The purpose of this study was to evaluate the behavior of ovine meniscal cells seeded on biomaterials made from collagen and hyaluronan, respectively. Ovine meniscal cells were isolated from the medial menisci of stifle joints, expanded in monolayer culture, and seeded on scaffolds made of collagen type II and I/III and a hyaluronan derivative (Hyaff-11). The samples were cultured for 12 h and 7, 14, 21, and 28 days. Histological analysis, electron microscopy, biochemical assays for glycosaminoglycans (GAGs) and DNA, and reverse transcriptase polymerase chain reaction analysis for collagens were performed. The cells attached well to both biomaterials and produced tissue-specific proteins, such as GAG and collagen type I, over a period of 28 days. Differences between the biomaterials were seen with respect to cell distribution, cell morphology, and the dynamics of GAG synthesis. The results show that ovine meniscal cells express their phenotype in both biomaterials. In terms of biology, collagen and hyaluronan are both suitable for tissue engineering in meniscal regeneration. It remains to be determined which scaffold possesses adequate biomechanical properties for successful in vivo application.

The effect of age on the structure and composition of rat tendon fibrocartilage.

Biochemical and morphological aspects of fibrocartilages of calcaneal and deep digital flexor tendons in rats aged 30, 180 and 730 days were analyzed. In both tendons a stronger staining with Alcian blue, indicating the presence of proteoglycans, was detected in rats of 30 and 180 days. In animals 730 days old, it was restricted to the pericellular area. Ultrastructural analysis showed a more prominent pericellular matrix in calcaneal tendon compared to the deep digital flexor tendon. The biochemical analysis showed higher levels of proteins and glycosaminoglycans in the calcaneal tendon of 30-day-old rats compared to older rats. In the deep digital flexor tendon, no significant differences were observed between ages. The small proteoglycan, fibromodulin, was detected in both tendons of all ages, but in young rats it appeared to be running as a 210 kDa component, probably due to the association with collagen chains or self-association.

Topographical guidance of intervertebral disc cell growth in vitro: towards the development of tissue repair strategies for the anulus fibrosus.

The anulus fibrosus (AF) of the intervertebral disc consists of concentric sheets of collagenous matrix that is synthesised during embryogenesis by aligned disc cells. This highly organised structure may be severely disrupted during disc degeneration and/or herniation. Cell scaffolds that incorporate topographical cues as contact guidance have been used successfully to promote the healing of injured tendons. Therefore, we have investigated the effects of topography on disc cell growth. We show that disc cells from the AF and nucleus pulposus (NP) behaved differently in monolayer culture on micro-grooved membranes of polycaprolactone (PCL). Both cell types aligned to and migrated along the membrane's micro-grooves and ridges, but AF cells were smaller (or less spread), more bipolar and better aligned to the micro-grooves than NP cells. In addition, AF cells were markedly more immunopositive for type I collagen, but less immunopositive for chondroitin-6-sulphated proteoglycans than NP cells. There was no evidence of extracellular matrix (ECM) deposition. Disc cells cultured on non-grooved PCL did not show any preferential alignment at sub-confluence and did not differ in their pattern of immunopositivity to those on grooved PCL. We conclude that substratum topography is effective in aligning disc cell growth and may be useful in tissue engineering for the AF. However, there is a need to optimise cell sources and/or environmental conditions (e.g. mechanical influences) to promote the synthesis of an aligned ECM.

Light microscopic, immunohistochemical, and ultrastructural findings in congenital fibular aplasia or hypoplasia (FAH).

Congenital aplasia or hypoplasia of the fibula (FAH) is a rare malformation that is defined by a partial or complete absence of the fibular bone. Etiology and pathogenesis are unknown and the precise morphology of the tissue cord replacing the malformed fibula has not been well described. Therefore, tissue cord was examined in 8 patients with FAH. Light microscopic, immunohistochemical, and electron microscopic investigations showed a core of embryonic cartilage with collagen II and VI expressions surrounded by connective tissue. Although collagen II expression is typical for chondroid differentiation, collagen VI reactivity is normally seen in articular cartilage and tendon-like fibrocartilaginous tissue but is absent in hyaline cartilage. Further ultrastructural analyses by electron microscopy supported these findings. The histomorphologic changes correspond to the histologic findings of Papenbrock et al. (2000, Mech Dev 92:113-123) who produced a congenital malformation in transgenic mice that resembled FAH by overexpression of Hox c11.