Histological and molecular characterization of the growing nasal septum in mice.

The nasal septum is a cartilaginous structure that serves as a pacemaker for the development of the midface. The septum is a hyaline cartilage which is surrounded by a perichondrium and epithelium. It remains cartilaginous anteriorly, but posteriorly it undergoes endochondral ossification to form the perpendicular plate of the ethmoid. Understanding of hyaline cartilage differentiation stems predominantly from investigations of growth plate cartilage. It is currently unclear if the morphological and molecular properties of the differentiating nasal septum align with what is known from the growth plate. In this study, we describe growth, molecular, and cellular characteristics of the nasal septum with reference to hyaline cartilage differentiation. The nasal septum grows asynchronous across its length with phases of rapid growth interrupted by more stagnant growth. Growth appears to be driven predominantly by acquisition of chondrocyte hypertrophy. Similarly, cellular differentiation is asynchronous, and differentiation observed in the anterior part precedes posterior differentiation. Overall, the nasal septum is structurally and molecularly heterogeneous. Early and extensive chondrocyte hypertrophy but no ossification is observed in the anterior septum. Onset of hypertrophic chondrocyte differentiation coincided with collagen fiber deposition along the perichondrium. Sox9, Col2, Col10, Mmp13, Sp7, and Runx2 expression was heterogeneous and did not always follow the expected pattern established from chondrocyte differentiation in the growth plate. The presence of hypertrophic chondrocytes expressing bone-related proteins early on in regions where the nasal septum does not ossify displays incongruities with current understanding of hyaline cartilage differentiation. Runx2, Collagen II, Collagen X, and Sp7 commonly used to mark distinct stages of chondrocyte maturation and early bone formation show wider expression than expected and do not align with expected cellular characteristics. Thus, the hyaline cartilage of the nasal septum is quite distinct from growth plate hyaline cartilage, and caution should be taken before assigning cartilage properties to less well-defined cartilage structures using these commonly used markers. Beyond the structural description of the nasal cartilage, this study also provides important information for cartilage tissue engineering when using nasal septal cartilage for tissue regeneration.

Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-ß, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-ß2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.

Cultivo condral in vitro bajo estímulos de compresión y cizallamiento. De las células troncales mesenquimales al cartílago hialino / In vitro chondral culture under compression and shear stimuli. From mesenchymal stem cells to hyaline cartilage

INTRODUCCIÓN: La creación in vitro de cartílago hialino articular supone un reto, ya que, a día de hoy, no se ha conseguido la síntesis ex vivo de un tejido estructurado con las mismas propiedades biomecánicas e histológicas del cartílago articular. Para simular las condiciones fisiológicas hemos diseñado un sistema de cultivo in vitro que reproduce el movimiento articular. MATERIAL Y MÉTODO: Hemos desarrollado un biorreactor de cultivo celular que imprime un estímulo mecánico sobre una matriz de elastina en la que están embebidas células troncales mesenquimales (MSC). La primera fase de estudio corresponde al desarrollo de un biorreactor para cultivo de cartílago hialino y la comprobación de la viabilidad celular en la matriz de elastina en ausencia de estímulo. La segunda fase del estudio engloba el cultivo de MSC bajo estímulo mecánico y el análisis del tejido resultante. RESULTADOS: Tras el cultivo bajo estímulo mecánico no obtuvimos tejido hialino por falta de celularidad y desestructuración de la matriz. CONCLUSIÓN: El patrón de estímulo utilizado no ha resultado efectivo para la generación de cartílago hialino, por lo que se deberán explorar otras combinaciones en futuras investigaciones

INTRODUCTION: The in vitro creation of hyaline joint cartilage is a challenge since, to date, the ex vivo synthesis of a structured tissue with the same biomechanical and histological properties of the joint cartilage has not been achieved. To simulate the physiological conditions we have designed an in vitro culture system that reproduces joint movement. MATERIAL AND METHOD: We have developed a cell culture bioreactor that prints a mechanical stimulus on an elastin matrix, in which mesenchymal stem cells (MSC) are embedded. The first phase of study corresponds to the development of a bioreactor for hyaline cartilage culture and the verification of cell viability in the elastin matrix in the absence of stimulus. The second phase of the study includes the MSC culture under mechanical stimulus and the analysis of the resulting tissue. RESULTS: After culture under mechanical stimulation we did not obtain hyaline tissue due to lack of cellularity and matrix destructuring. CONCLUSION: The stimulus pattern used has not been effective in generating hyaline cartilage, so other combinations should be explored in future research

Glucocorticoids induce osteoporosis mediated by glucocorticoid receptor-dependent and -independent pathways.

Clinically, glucocorticoids (GCs) are widely used to treat inflammation-related diseases; however, their long-term use causes side effects, such as osteoporosis and predisposition to bone fractures, known as glucocorticoid-induced osteoporosis (GIOP). Nr3c1 is the major glucocorticoid receptor, and its downstream signaling pathway is involved in regulating various intracellular physiological processes, including those related to bone cells; however, its mechanism in glucocorticoid-induced osteoporosis (GIOP) remains unclear. In this study, a zebrafish nr3c1-mutant was successfully generated using CRISPR/Cas9 technology to investigate the role of nr3c1 in GIOP. Mutations in nr3c1 altered cartilage development and significantly decreased bone mineralization area. Additionally, qRT-PCR results showed that the expression of extracellular matrix-, osteoblast-, and osteoclast-related genes was altered in the nr3c1-mutant. The GC-Nr3c1 pathway regulates the expression of extracellular matrix-, osteoblast-, and osteoclast-related genes via Nr3c1-dependent and Nr3c1-independent pathways. A dual-luciferase reporter assay further revealed that GCs and Nr3c1 transcriptionally regulate matrix metalloproteinase 9 (mmp9), alkaline phosphatase (alp), and acid phosphatase 5a (acp5a). This study reveals that GCs/Nr3c1 affect the expression of genes involved in bone metabolism and provides a basis to determine the role of GIOP and Nr3c1 in bone metabolism and development. We also identified a new effector target for the clinical treatment of GIOP.

Maternal HMB treatment affects bone and hyaline cartilage development in their weaned piglets via the leptin/osteoprotegerin system.

It has been demonstrated in animal studies that prenatal administration of ß-hydroxy-ß-methylbutyrate (HMB, metabolite of leucine) influences general growth and mechanical endurance of long bones in newborn offspring in sex-dependent manner. The present experiment was conducted to evaluate the effect of HMB treatment of pregnant sows on bone development in offspring at weaning. From 70th day until the 90th day of gestation, sows received either a basal diet (n = 12) or the same diet supplemented with HMB (n = 12) at the dose of 0.2 g/kg of body weight/day. Femora obtained from six males and females in each group weaned at the age of 35 days were examined. Maternal HMB treatment significantly enhanced body weight and changed bone morphology increasing femur mechanical strength in both sexes. Maternal HMB supplementation also elevated bone micro- and macroelement concentrations and enhanced content of proteoglycans in articular cartilage. Based on the obtained results, it can be concluded that maternal HMB supplementation in the mid-gestation period significantly accelerated bone development in both sexes by upregulation of a multifactorial system including leptin and osteoprotegerin. However, the sex (irrespective of the HMB treatment) was the factor which influenced the collagen structure in cartilages and trabecular bone, as demonstrated both by the Picrosirius red staining and performed analysis of thermal stability of collagenous tissues. The structural differences in collagen between males and females were presumably related to a different collagen maturity. No studies conducted so far provided a detailed morphological analysis of bone, articular cartilage, growth plate and the activities of the somatotropic and pituitary-gonadal axes, as well as leptin/osteoprotegerin system in weaned offspring prenatally treated with HMB. This study showed also the relationship between the maternal HMB treatment and bone osteometric and mechanical traits, hormones, and growth and bone turnover markers such as leptin, osteoprotegerin and insulin-like growth factor-1.

Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with Organically Complexed Copper in a Cu-Deficient Diet.

Tibial mechanical, chemical, and histomorphometrical traits were investigated for growing male Ross 308 broiler chickens fed diets that had copper (Cu) from organic source at a lowered level of 25% of the daily requirement (4 mg kg-1 of a premix) with or without phytase. Dietary treatments were control non-copper, non-phytase group (0 Suppl); 4 mg kg-1 Cu non-phytase group (25%Cu); and 4 mg kg-1 Cu + 500 FTU kg-1 phytase group (25%Cu + phyt). The results show that birds fed with the addition of phytase exhibited improved weight gain and final body weight and had increased serum IGF-1 and osteocalcin concentrations. The serum concentration of Cu and P did not differ between groups; however, Ca concentration decreased in the 25%Cu + phyt group when compared to the 25%Cu group. Added Cu increased bone Ca, P, Cu, and ash content in Cu-supplemented groups, but bone weight and length increased only by the addition of phytase. Bone geometry, yield, and ultimate strengths were affected by Cu and phytase addition. A decrease of the elastic stress and ultimate stress of the tibia in Cu-supplemented groups was observed. The histomorphometric analysis showed a positive effect of Cu supplementation on real bone volume and trabecular thickness in the tibia metaphyseal trabeculae; additionally, phytase increased the trabeculea number. The supplementation with Cu significantly increased the total articular cartilage and growth plate cartilage thickness; however, the changes in thickness of particular zones were dependent upon phytase addition. In summary, dietary Cu supplements given to growing broilers with Cu in their diet restricted to 25% of the daily requirement had a positive effect on bone metabolism, and phytase supplementation additionally improved cartilage development.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

The "Mechanostat Theory" of Frost and the OPG/RANKL/RANK System.

Frost's great interest to elucidate the principles of action underlying skeletal deformities, during, and after growth, urged him to undertake an extensive study of the mammalian skeleton. He suggested that survival of the skeleton (but also of other tissues, such as fibrous tissue, hyaline cartilage, fibrocartilage, cementum, or dentin) requires the functional coordination of modeling and remodeling. Modeling adapts bone to overloads, by enhancing additions of new bone and by changing bone architecture, and remodeling adapts bone to underloads by removing bone next to marrow and conserving normally used bone. There exists a mechanism that monitors bone metabolism (longitudinal growth, bone modeling, and remodeling activities) in relation to mechanical usage, the "mechanostat." Recent literature has presented new information regarding the physiological procedure of osteoclast and osteoblast activation. It has been understood that the OPG/RANKL/RANK proteinic system regulates bone metabolism by exerting biological effects on osteoblasts or osteoclasts. The same proteinic network, also regulates alveolar remodeling during tooth movement, as well as physiological root resorption and root resorption during orthodontic tooth movement. The aim of the present review is the presentation and evaluation of recent information in the field of osteoclast and osteoblast biology, as regards to the "mechanostat theory" of Frost. An attempt will be made to elucidate, whether recent data can support this remarkable theory and reveal the biological mechanisms behind it.

Self-crosslinked oxidized alginate/gelatin hydrogel as injectable, adhesive biomimetic scaffolds for cartilage regeneration.

Biopolymeric hydrogels that mimic the properties of extracellular matrix have great potential in promoting cellular migration and proliferation for tissue regeneration. The authors reported earlier that rapidly gelling, biodegradable, injectable hydrogels can be prepared by self-crosslinking of periodate oxidized alginate and gelatin in the presence of borax, without using any toxic crosslinking agents. The present paper investigates the suitability of this hydrogel as a minimally invasive injectable, cell-attractive and adhesive scaffold for cartilage tissue engineering for the treatment of osteoarthritis. Time and frequency sweep rheology analysis confirmed gel formation within 20s. The hydrogel integrated well with the cartilage tissue, with a burst pressure of 70±3mmHg, indicating its adhesive nature. Hydrogel induced negligible inflammatory and oxidative stress responses, a prerequisite for the management and treatment of osteoarthritis. Scanning electron microscopy images of primary murine chondrocytes encapsulated within the matrix revealed attachment of cells onto the hydrogel matrix. Chondrocytes demonstrated viability, proliferation and migration within the matrix, while maintaining their phenotype, as seen by expression of collagen type II and aggrecan, and functionality, as seen by enhanced glycosoaminoglycan (GAG) deposition with time. DNA content and GAG deposition of chondrocytes within the matrix can be tuned by incorporation of bioactive signaling molecules such as dexamethasone, chondroitin sulphate, platelet derived growth factor (PDGF-BB) and combination of these three agents. The results suggest that self-crosslinked oxidized alginate/gelatin hydrogel may be a promising injectable, cell-attracting adhesive matrix for neo-cartilage formation in the management and treatment of osteoarthritis.

Isolation and ex vivo expansion of synovial mesenchymal stromal cells for cartilage repair.

BACKGROUND AIMS: Hyaline articular cartilage is a highly specialized tissue that offers a low-friction and wear-resistant interface for weight-bearing surface articulation in diarthrodial joints, but it lacks vascularity. It displays an inherent inability to heal when injured in a skeletally mature individual. Joint-preserving treatment procedures such as mosaicplasty, débridement, perichondrium transplantation and autologous chondrocyte implantation have shown variable results, and the average long-term result is sub-standard. Because of these limitations of the treatment methods and lack of intrinsic repair capacity of mature cartilage tissue, an alternative treatment approach is needed, and synovial mesenchymal stromal cells (SMSCs) represent an attractive therapeutic alternative because of their ex vivo proliferation capacity, multipotency and ability to undergo chondrogenesis. METHODS: SMSCs were isolated from tissues obtained by arthroscopy using two types of biopsies. Ex vivo cell expansion was accomplished under static and dynamic culture followed by characterization of cells according to the International Society for Cellular Therapy guidelines. Kinetic growth models and metabolite analysis were used for understanding the growth profile of these cells. RESULTS: For the first time, SMSCs were expanded in stirred bioreactors and achieved higher cell density in a shorter period of time compared with static culture or with other mesenchymal stromal cell sources. CONCLUSIONS: In this study we were able to achieve (8.8 ± 0.2) × 10(5) cells within <2 weeks in dynamic culture under complete xeno-free conditions. Our results also provided evidence that after dynamic culture these cells had an up-regulation of chondrogenic genes, which can be a potential factor for articular cartilage regeneration in clinical settings.

Evaluation of the effects of electrical stimulation on cartilage repair in adult male rats.

This study describes the organization of mature hyaline xiphoid cartilage during repair in animals submitted to electrical current stimulation. Twenty male Wistar rats, 90 days old, were divided into a control group (CG) and a treated group (TG). A cylindrical full-thickness cartilage defects were created with a 3-mm punch in anesthetized animals. After 24h, TG received daily applications of a continuous electrical current (1Hz/20µA) for 5min. The animals were sacrificed after 7, 21 and 35 days for structural analysis. In CG, the repair tissue presented fibrous characteristics, with fibroblastic cells being infiltrated and permeated by blood vessels. Basophilic foci of cartilage tissue were observed on day 35. In TG, the repair tissue also presented fibrous characteristics, but a larger number of thick collagen fibers were seen on day 21. A large number of cartilaginous nests were observed on day 35. Cell numbers were significantly higher in TG. Calcification points were detected in TG on day 35. There was no difference in elastic fibers between groups. Ultrastructural analysis revealed the presence of chondrocyte-like cells in CG at all time points, but only on days 21 and 35 in TG. The amount of cuprolinic blue-stained proteoglycans was higher in TG on day 35. Microcurrent stimulation accelerates the repair process in non-articular hyaline cartilage.

Bone morphogenetic protein-7 promotes chondrogenesis in human amniotic epithelial cells.

Bone morphogenetic proteins (BMPs) play important roles at multiple stages of chondrogenesis. This study was undertaken to investigate the potential role of bone morphogenetic protein-7 (BMP-7) in the differentiation of chondrocytes using tissue engineering techniques. The impact of BMP-7 on human amniotic epithelial cells (hAECs) was tested. The hAECs were treated either with recombinant human BMP-7 cDNA or with transforming growth factor beta 1 (TGF-ß1) as a positive control for three weeks in vitro. Cartilaginous differentiation and proliferation were assayed by quantitative RT-PCR, histology, and in situ hybridization. Our results were such that hAECs treated with either BMP-7 or TGF-ß1 expressed cartilage markers (aggrecan, Sox9, CEP-68, and type II and X collagens) within three weeks. Compared with a control vector, BMP-7 induced a decrease in type I collagen expression, while the transcription of the cartilage-specific type II collagen remained stable. In induction experiments, BMP-7 transgenic hAECs exhibited the largest amount of matrix synthesis. In conclusion, these data indicate that BMP-7 plays an important role in inducing the production of cartilage by hAECs in vitro. Cartilage differentiation and matrix maturation can be promoted by BMPs in a cartilage engineering paradigm. These properties make BMPs promising tools in the engineering of cartilaginous joint bio-prostheses and as candidate biological agents or genes for cartilage stabilisation.

Effects of different levels of protein intake and physical training on growth and nutritional status of young rats.

This study aimed to investigate the effects of physical training, and different levels of protein intake in the diet, on the growth and nutritional status of growing rats. Newly-weaned Wistar rats (n=48) were distributed into six experimental groups; three of them were subjected to physical swim training (1 h per day, 5 d per week, for 4 wk, after 2 wk of familiarization) and the other three were considered as controls (non-trained). Each pair of groups, trained and non-trained, received diets with a different level of protein in their composition: 14%, 21% or 28%. The animals were euthanized at the end of the training period and the following analyses were performed: proteoglycan synthesis as a biomarker of bone and cartilage growth, IGF-I (insulin-like growth factor-I) assay as a biomarker of growth and nutritional status, total RNA and protein concentration and protein synthesis measured in vivo using a large-dose phenylalanine method. As a main finding, increased dietary protein, combined with physical training, was able to improve neither tissue protein synthesis nor muscle growth. In addition, cartilage and bone growth seem to be deteriorated by the lower and the higher levels of protein intake. Our data allow us to conclude that protein enhancement in the diet, combined with physical exercise, does not stimulate tissue protein synthesis or muscle mass growth. Furthermore, physical training, combined with low protein intake, was not favorable to bone development in growing animals.

Regulation of the chondrogenic phenotype in culture.

In recent years, there has been a great deal of interest in the development of regenerative approaches to produce hyaline cartilage ex vivo that can be utilized for the repair or replacement of damaged or diseased tissue. It is clinically imperative that cartilage engineered in vitro mimics the molecular composition and organization of and exhibits biomechanical properties similar to persistent hyaline cartilage in vivo. Experimentally, much of our current knowledge pertaining to the regulation of cartilage formation, or chondrogenesis, has been acquired in vitro utilizing high-density cultures of undifferentiated chondroprogenitor cells stimulated to differentiate into chondrocytes. In this review, we describe the extracellular matrix molecules, nuclear transcription factors, cytoplasmic protein kinases, cytoskeletal components, and plasma membrane receptors that characterize cells undergoing chondrogenesis in vitro and regulate the progression of these cells through the chondrogenic differentiation program. We also provide an extensive list of growth factors and other extracellular signaling molecules, as well as chromatin remodeling proteins such as histone deacetylases, known to regulate chondrogenic differentiation in culture. In addition, we selectively highlight experiments that demonstrate how an understanding of normal hyaline cartilage formation can lead to the development of novel cartilage tissue engineering strategies. Finally, we present directions for future studies that may yield information applicable to the in vitro generation of hyaline cartilage that more closely resembles native tissue.

MR imaging insights into skeletal maturation: what is normal?

Skeletal growth and maturation in children is a dynamic process that can be documented with magnetic resonance (MR) imaging. There are predictable normal developmental changes that must be differentiated from pathologic processes. This review discusses the histologic structure and MR imaging appearance of normal development-related changes of the musculoskeletal system in children, including those that may be mistaken for abnormalities.

Crushed bone grafts and a collagen membrane are not suitable for enhancing cartilage quality in the regeneration of osteochondral defects--an in vivo study in sheep.

Hyaline joint cartilage has only a limited potential for self-repair. Some of the published techniques for osteochondral defect therapy try to improve that potential. In this study, it was hypothesised that one of those surgical techniques, the crushed transplanted bone graft together with a collagen membrane, accelerates significantly the reconstruction of the subchondral bone plate and improves the mechanical and histological quality of repaired cartilage in osteochondral defects compared to an empty control defect. In order to test this hypothesis, defects were created in the left knee of 12 sheep and filled either with autologous crushed bone graft or left empty. The animals were sacrificed after 3 (n = 6) and 6 (n = 6) months. No differences were found either macroscopically or histomorphometrically between the bone graft and empty control defects. The biomechanical as well as the histological results of the bone graft defects were inferior to the control defects with inflammatory processes caused either by bone graft or membrane remnants. Based on the results in this sheep model, the filling of subchondral bone defects with compacted cancellous bone should be carefully reconsidered.

Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation.

Parathyroid hormone (PTH; 10(-7) to 10(-15) M) decreased terminal chondrogenesis in the avian sterna. During the first half of an 8-day culture, 100 nM PTH (1-34) significantly increased sternal length and downregulated the deposition of type X collagen and its mRNA expression. However, it remains unclear how PTH increased cartilaginous growth. In this study, we examined growth by both cell proliferation and analysis of cyclin d1 and collagen mRNA. Types II, IX, and X collagens and cyclin d1 mRNA were quantified through real-time RT-PCR, while Ki-67 was used as an immunohistochemical proliferation marker. Extracellular matrix content was measured through mRNA quantification of types II, IX, and X collagen and observing deposition of the same collagens. PTH significantly increased the proliferation marker Ki-67 in the sternal cephalic region. There was less type II and X collagen in PTH-treated sterna with concomitant decreases in mRNA production, suggesting that proliferation was the major contributor to cartilage growth in the presence of PTH/PTH-related peptide receptor activation. In conclusion, these experiments demonstrated that PTH increased cartilage growth by upregulating cell proliferation or other extracellular matrix components.